Tetracycline compositions

ABSTRACT

The present invention relates to compositions, pharmaceutical compositions, and methods for preparing the same, comprising a tetracycline with improved stability and solubility. Some embodiments include a tetracycline with an excess of a divalent or trivalent cation.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/654,018, filed Oct. 17, 2012, which is a continuation ofInternational Application No. PCT/US2011/036351 filed on May 12, 2011,which claims priority to U.S. Provisional Application No. 61/392,304filed Oct. 12, 2010, and to U.S. Provisional Application No. 61/334,106filed May 12, 2010, the contents of which are incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates to tetracycline compositions and methodsfor preparing and using the same. Some embodiments include atetracycline with an excess of a divalent or trivalent cation.

BACKGROUND OF THE INVENTION

Tetracyclines are used as broad spectrum antibiotics to treat variousbacterial infections, such as infections of the respiratory tract,sinuses, middle ear, urinary tract, and intestines, and can be used inthe treatment of gonorrhoea, especially in patients allergic toβ-lactams and macrolides. Tetracyclines interfere with the proteinsynthesis of Gram positive and Gram-negative bacteria by preventing thebinding of aminoacyl-tRNA to the ribosome. The action of tetracyclinesis bacteriostatic (preventing growth of bacteria) rather than killing(bactericidal).

Tetracyclines degrade rapidly to form epitetracycline,anhydrotetracycline, epianhydrotetracycline, and other degradationproducts. Once degraded, tetracyclines have small therapeutic value,since the degradation products have no therapeutically useful activity.Degradation begins as soon as the antiobiotic is in solution, andcontinues until reaching an equilibrium of antibiotic and epimerconcentrations. The equilibrium point is temperature and pH dependent,with more epimer being formed at higher temperatures and lower pH.Oxidation and other side reactions cause further degradation. Thus,tetracyclines can have a limited existence in aqueous environments intheir active form. Moreover, the degradation products of tetracyclinesare toxic and can cause Fanconi syndrome, a potentially fatal diseaseaffecting proximal tubular function in the nephrons of the kidneys.

There is a need to provide hospital staff with the flexibility andadvantages that come with longer admixture and reconstitution timeswithout the need for refrigeration so that for instance, a hospitalpharmacist could prepare a solution the day before it is needed.Furthermore, often after a natural disaster such as hurricanes,earthquakes, or tsunamis, access to refrigeration equipment can bescarce and may be further impeded by the lack of electricity. Stableformulations of tetracyclines could be stored as a solution, negatingthe need for reconstitution, and allowing its use in inhalers ornebulizers for outpatient use.

In addition, some tetracyclines can cause tetracycline-inducedhemolysis. This hemolysis can lead to venous phlebitis at the site ofinjection when administered intravenously, resulting in irritation andpotentially limiting the volumes of infusion that can be tolerated.Thus, there is a need for formulations of such tetracyclines that reducethe incidence of hemolysis.

SUMMARY OF THE INVENTION

The present invention relates to tetracycline compositions and methodsfor preparing and using the same. Some embodiments include atetracycline with an excess of a divalent or trivalent cation.

Some embodiments include pharmaceutical compositions. In someembodiments the pharmaceutical compositions comprise an aqueous solutionof minocycline and a divalent or trivalent cation, wherein the molarratio of divalent or trivalent cation to minocycline is greater than 2:1and wherein the solution does not comprise a pharmaceutically acceptableoil and is suitable for intravenous administration.

In some embodiments the pharmaceutical compositions comprise an aqueoussolution of minocycline and a divalent or trivalent cation, wherein themolar ratio of divalent or trivalent cation to minocycline is greaterthan 2:1 and wherein the solution has a pH greater than 4 and less than5 and is suitable for intravenous administration.

In some embodiments the pharmaceutical compositions comprise an aqueoussolution of a 7-dimethylamino-tetracycline antibiotic and a divalent ortrivalent cation, wherein the molar ratio of divalent or trivalentcation to 7-dimethylamino-tetracycline antibiotic is greater than 3:1and wherein the solution does not comprise a pharmaceutically acceptableoil, gluconate, or a pyridine-containing compound, has a pH greater than2 and less than 7, and is suitable for intravenous administration.

In some embodiments, the solution does not comprise polyoxyethylenehydrogenated castor oil.

In some embodiments, the solution does not comprise an antioxidant.

In some embodiments, the solution does not comprise apyridine-containing compound.

In some embodiments, the solution does not comprise nicotinamide.

In some embodiments, the solution does not comprise an alcohol.

In some embodiments, the solution does not comprise glycerol.

In some embodiments, the solution does not comprise polyethylene glycol.

In some embodiments, the solution does not comprise gluconate.

In some embodiments, the solution does not comprise a pyrrolidonecompound.

In some embodiments, the solution does not comprise a water-misciblelocal anaesthetic.

In some embodiments, the water-miscible local anaesthetic is procaine.

In some embodiments, the solution does not comprise urea.

In some embodiments, the solution does not comprise lactose.

In some embodiments, the solution does not comprise a dehydrating agent.In some embodiments, the dehydrating agent is selected from the groupconsisting of ethyl acetate, acetic anhydride, absolute ethanol, ethylacetate, acetic anhydride, and mixtures thereof.

In some embodiments, the solution has a pH of less than 7. In someembodiments, the solution has a pH of less than 6. In some embodiments,the solution has a pH of less than 5.

In some embodiments, the solution has a pH greater than 2 and less than7. In some embodiments, the solution has a pH greater than 4 and lessthan 7. In some embodiments, the solution has a pH greater than 4 andless than 6. In some embodiments, the solution has a pH greater than 4and less than 5.

In some embodiments, the molar ratio of divalent or trivalent cation tominocycline is greater than 3:1. In some embodiments, the molar ratio ofdivalent or trivalent cation to minocycline is greater than 5:1. In someembodiments, the molar ratio of divalent or trivalent cation tominocycline is greater than 8:1. In some embodiments, the molar ratio ofdivalent or trivalent cation to minocycline is greater than 10:1.

In some embodiments, the osmolality of the solution is less than 500mOsm/kg. In some embodiments, the osmolality of the solution is lessthan 400 mOsm/kg. In some embodiments, the osmolality of the solution isless than 350 mOsm/kg.

In some embodiments, the concentration of minocycline is at least 1mg/ml. In some embodiments, the concentration of minocycline is at least5 mg/ml. In some embodiments, the concentration of minocycline is atleast 10 mg/ml.

In some embodiments, the solution comprises magnesium sulfate. In someembodiments, the solution comprises magnesium oxide. In someembodiments, the solution comprises magnesium acetate. In someembodiments, the solution comprises magnesium chloride.

In some embodiments, the solution comprises a buffer. In someembodiments, the solution comprises acetate.

In some embodiments, the solution comprises a base. In some embodiments,the base comprises NaOH.

In some embodiments, the cation is selected from iron, copper, zinc,manganese, nickel, cobalt, aluminum, calcium, magnesium and gallium. Insome embodiments, the cation is selected from magnesium, calcium, andzinc. In some embodiments, the cation is magnesium.

In some embodiments, the 7-dimethylamino-tetracycline is selected fromminocycline, PTK796, and a glycylcycline. In some embodiments, theglycylcycline is tigecycline. In some embodiments, the7-dimethylamino-tetracycline is minocycline. In some embodiments, the7-dimethylamino-tetracycline is PTK796.

Some embodiments include pharmaceutical compositions comprising 10 mg/mlminocycline, MgCl₂, and NaOH, wherein the Mg to minocycline molar ratiois 5:1, and the pH is greater than 4.5 and less than 5.5.

Some embodiments include pharmaceutical compositions comprising 10 mg/mlminocycline, MgSO₄, and sodium acetate, wherein the Mg to minocyclinemolar ratio is 5:1, the pH is greater than 4.5 and less than 5.5, andthe osmolality is greater than 275 mOsm/kg and less than 375 mOsm/kg.

Some embodiments include pharmaceutical compositions comprising 10 mg/mlminocycline and Mg(C₂H₃O₂)₂, wherein the Mg to minocycline molar ratiois 5:1, and the pH is greater than 4.5 and less than 5.5.

Some embodiments include pharmaceutical compositions comprising 10 mg/mlminocycline, MgSO₄, and NaOH, wherein the Mg to minocycline molar ratiois 5:1, the pH is greater than 4.5 and less than 5.5, and the osmolalityis greater than 150 mOsm/kg and less than 250 mOsm/kg.

Some embodiments include pharmaceutical compositions comprising 5 mg/mltigecycline, MgSO₄, and NaOH, wherein the Mg to tigecycline molar ratiois 5:1, and the pH is greater than 5.5 and less than 6.5.

Some embodiments include pharmaceutical compositions comprising 5 mg/mltigecycline, MgSO₄, and NaOH, wherein the Mg to tigecycline molar ratiois 12:1, and the pH is greater than 5.5 and less than 6.5.

Some embodiments include pharmaceutical compositions comprising 5 mg/mltigecycline, MgCl₂, and NaOH, wherein the Mg to tigecycline molar ratiois 5:1, and the pH is greater than 5.5 and less than 6.5.

Some embodiments include pharmaceutical compositions comprising 5 mg/mltigecycline, MgCl₂, and NaOH, wherein the Mg to tigecycline molar ratiois 12:1, and the pH is greater than 5.5 and less than 6.5.

Some embodiments include pharmaceutical compositions suitable fortopical administration comprising 5 mg/ml tigecycline, MgSO₄, and NaOH,wherein the Mg to tigecycline molar ratio is 5:1, and the pH is greaterthan 6.0 and less than 7.0.

Some embodiments include pharmaceutical compositions suitable fortopical administration comprising 5 mg/ml tigecycline, MgSO₄, and NaOH,wherein the Mg to tigecycline molar ratio is 12:1, and the pH is greaterthan 6.0 and less than 7.0.

Some embodiments include pharmaceutical compositions suitable fortopical administration comprising 5 mg/ml tigecycline, CaCl₂, and NaOH,wherein the Ca to tigecycline molar ratio is 5:1, and the pH is greaterthan 6.0 and less than 7.0.

Some embodiments include pharmaceutical compositions suitable fortopical administration comprising 5 mg/ml tigecycline, CaCl₂, and NaOH,wherein the Ca to tigecycline molar ratio is 12:1, and the pH is greaterthan 6.0 and less than 7.0.

Some embodiments include water-soluble solid compositions comprisingminocycline or a salt thereof and a salt that comprises a divalent ortrivalent cation.

Some embodiments include water-soluble solid compositions comprising a7-dimethylamino-tetracycline antibiotic or a salt thereof and a saltcomprising a divalent or trivalent cation, wherein the molar ratio ofdivalent or trivalent cation to 7-dimethylamino-tetracycline antibioticis greater than 3:1 and wherein the composition does not comprisegluconate or a pyridine-containing compound.

In some embodiments, the molar ratio of divalent or trivalent cation tominocycline is greater than 1:1. In some embodiments, the molar ratio ofdivalent or trivalent cation to minocycline is greater than 2:1. In someembodiments, the molar ratio of divalent or trivalent cation tominocycline is greater than 3:1. In some embodiments, the molar ratio ofdivalent or trivalent cation to the minocycline or the7-dimethylamino-tetracycline antibiotic is greater than 5:1. In someembodiments, the molar ratio of divalent or trivalent cation to theminocycline or the 7-dimethylamino-tetracycline antibiotic is at greaterthan 8:1. In some embodiments, the molar ratio of divalent or trivalentcation to the minocycline or the 7-dimethylamino-tetracycline antibioticis greater than 10:1.

Some embodiments include compositions in the form of a lyophile.

In some embodiments, the salt is magnesium sulfate.

In some embodiments, the salt is calcium chloride.

In some embodiments, the composition comprises sodium acetate.

In some embodiments, the composition comprises NaOH.

In some embodiments, the salt is selected from magnesium chloride,magnesium bromide, magnesium sulfate, calcium chloride, calcium bromide,calcium sulfate, zinc chloride, gallium chloride, magnesium malate,magnesium citrate, magnesium acetate, calcium citrate, zinc acetate, andzinc citrate.

In some embodiments, the composition does not comprise an antioxidant.

In some embodiments, the composition does not comprise apyridine-containing compound. In some embodiments, the composition doesnot comprise nicotinamide.

In some embodiments, the composition does not comprise gluconate.

In some embodiments, the 7-dimethylamino-tetracycline is selected fromminocycline, PTK796, and a glycylcycline. In some embodiments, theglycylcycline is tigecycline. In some embodiments, the7-dimethylamino-tetracycline is minocycline. In some embodiments, the7-dimethylamino-tetracycline is PTK796.

Some embodiments include methods for preparing a pharmaceuticalcomposition comprising dissolving the water-soluble solid composition ofany one of the water-soluble solid compositions provided herein in waterto form a solution

Some embodiments include methods for preparing a pharmaceuticalcomposition comprising dissolving a 7-dimethylamino-tetracycline in asolution comprising a divalent or trivalent cation.

Some embodiments include methods for preparing a pharmaceuticalcomposition comprising dissolving a 7-dimethylamino-tetracycline in asolution comprising a divalent or trivalent cation; adjusting the pH ofthe solution; and lyophilizing the composition.

In some embodiments, the 7-dimethylamino-tetracycline is selected fromminocycline, PTK796, and a glycylcycline. In some embodiments, theglycylcycline is tigecycline.

In some embodiments, the pH of the solution is adjusted to be less than6. In some embodiments, the pH of the solution is adjusted to be lessthan 5.

In some embodiments, the pH of the solution is adjusted to be greaterthan 2 and less than 7. In some embodiments, the pH of the solution isadjusted to be greater than 4 and less than 7. In some embodiments, thepH of the solution is adjusted to be greater than 4 and less than 6. Insome embodiments, the pH of the solution is adjusted to be greater than4 and less than 5.

In some embodiments, adjusting the pH comprises adding an acid. In someembodiments, the acid is HCl.

In some embodiments, adjusting the pH comprises adding a base. In someembodiments, the base is NaOH.

In some embodiments, adjusting the pH comprises forming a buffer. Insome embodiments, forming the buffer comprises adding sodium acetate.

In some embodiments, the divalent or trivalent cation is selected fromiron, copper, zinc, manganese, nickel, cobalt, aluminum, calcium,magnesium and gallium. In some embodiments, the cation is selected frommagnesium, calcium, and zinc. In some embodiments, the cation ismagnesium.

Some embodiments include kits comprising a first container comprising adiluent that comprises an aqueous solution of a divalent or trivalentcation; and a second container comprising a solid composition soluble inthe diluent, wherein the solid composition comprises minocycline in anamount such that the molar ratio of the divalent or trivalent cation tominocycline is greater than 2:1.

Some embodiments include kits comprising a first container comprising adiluent that comprises an aqueous solution of a divalent or trivalentcation; and a second container comprising a solid composition soluble inthe diluent, wherein the solid composition comprises a7-dimethylamino-tetracycline antibiotic in an amount such that the molarratio of the divalent or trivalent cation to7-dimethylamino-tetracycline antibiotic is greater than 3:1.

In some embodiments, the diluent comprises an acid. In some embodiments,the acid is HCl.

In some embodiments, the diluent comprises a base. In some embodiments,the base is NaOH.

In some embodiments, the diluent comprises a buffer. In someembodiments, the diluent comprises sodium acetate.

In some embodiments, the pH of the diluent is greater than pH 6 and lessthan pH 8.

In some embodiments, the divalent or trivalent cation is selected fromiron, copper, zinc, manganese, nickel, cobalt, aluminum, calcium,magnesium and gallium. In some embodiments, the cation is selected frommagnesium, calcium, and zinc. In some embodiments, the cation ismagnesium.

In some embodiments, the molar ratio of divalent or trivalent cation tominocycline is greater than 3:1. In some embodiments, the molar ratio ofdivalent or trivalent cation to the minocycline or the7-dimethylamino-tetracycline antibiotic is greater than 5:1. In someembodiments, the molar ratio of divalent or trivalent cation to theminocycline or the 7-dimethylamino-tetracycline antibiotic is at greaterthan 8:1. In some embodiments, the molar ratio of divalent or trivalentcation to the minocycline or the 7-dimethylamino-tetracycline antibioticis greater than 10:1.

In some embodiments, the 7-dimethylamino-tetracycline is selected fromminocycline, PTK796, and a glycylcycline. In some embodiments, theglycylcycline is tigecycline. In some embodiments, the7-dimethylamino-tetracycline is minocycline. In some embodiments, the7-dimethylamino-tetracycline is PTK796.

Some embodiments include methods of treating or preventing a bacterialinfection in a subject, comprising administering the pharmaceuticalcomposition of any one of the pharmaceutical compositions providedherein to the subject via an intravenous route.

Some embodiments include methods of treating or preventing a bacterialinfection in a subject, comprising administering the pharmaceuticalcomposition made according to any one of the methods of preparing apharmaceutical compositions provided herein to the subject via anintravenous route.

In some embodiments, the intravenous administration includesadministering less than 200 ml of the composition.

In some embodiments, the intravenous administration includesadministering the composition in less than 60 minutes.

Some embodiments include methods of treating or preventing a bacterialinfection in a subject, comprising administering the pharmaceuticalcomposition of any one of the pharmaceutical compositions providedherein to the subject via a topical route.

Some embodiments include methods of treating or preventing a bacterialinfection in a subject, comprising administering the pharmaceuticalcomposition made according to any one of the methods of preparing apharmaceutical compositions provided herein to the subject via a topicalroute.

Some embodiments include compositions comprising tigecycline and adivalent or trivalent cation, wherein the molar ratio of said divalentor trivalent cation to said tigecycline is greater than 1:1.

In some embodiments, the tigecycline and divalent or trivalent cationare in aqueous solution.

In some embodiments, the molar ratio of said divalent or trivalentcation to said tigecycline is greater than 3:1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph of percent hemolysis of rabbit red blood cellsincubated with various concentrations of minocycline in varioussolutions relative to hemolysis in water, in which the minocyclinesolutions formulated with divalent cations were adjusted to ph 5.85.

FIG. 2 shows a graph of percent hemolysis of rabbit red blood cellsincubated with various concentrations of minocycline in varioussolutions relative to hemolysis in water.

FIG. 3 depicts a graph of rabbit RBC hemolysis caused by minocyclineformulated in different ratios of MgSO₄.

FIG. 4 depicts a graph of rabbit RBC hemolysis caused by minocyclineformulated in different ratios of MgCl₂.

FIG. 5 depicts a graph of rabbit RBC hemolysis caused by minocyclineformulated in different ratios of CaCl₂.

FIG. 6 depicts a graph for minocycline uptake by HVEC at variousconcentrations of divalent cation.

FIG. 7 depicts a graph for minocycline uptake by HVEC at variousconcentrations of divalent cation.

DETAILED DESCRIPTION

The present invention relates to tetracycline compositions and methodsfor preparing and using the same. Some embodiments include atetracycline with an excess of a metal cation. In some embodiments, thecompositions have improved stability against both oxidative degradationand epimerization. Some such compositions are therefore more stable whendissolved, lyophilized, reconstituted, and/or diluted than othercompositions. Some embodiments also provide compositions having a lowerlevel of tetracycline-induced hemolysis and resulting phlebitis.

It was unexpectedly discovered that the incidence oftetracycline-induced hemolysis can be greatly decreased by formulatingthe tetracycline with divalent or trivalent cations. In someembodiments, high molar ratios of divalent or trivalent cations totetracycline antibiotics significantly decreases hemolysis.

It was also unexpectedly discovered that the stability of aqueoussolutions of tetracyclines can be greatly increased by the addition ofdivalent or trivalent cations. In some embodiments, the stability ofaqueous solutions of tetracyclines increase with higher molar ratios ofdivalent or trivalent cations to tetracycline. Indeed, some suchsolutions were found to be stable for several weeks at 37° C.

In certain compositions, the solubility of a tetracycline antibiotic isdecreased in an aqueous solution comprising a multivalent cation. It hasbeen unexpectedly discovered that increasing the molar ratio ofmultivalent cation to such tetracycline antibiotics can increase thesolubility of the tetracycline. Accordingly, some embodiments describedherein provide solutions of a tetracycline with improved solubility.

Compositions

Some embodiments include compositions comprising a tetracyclineantibiotic or a salt thereof in combination with a divalent or trivalentcation. Tetracyclines include a family of structurally-related compoundsthat may have broad-spectrum antibiotic activities. Examples oftetracyclines include Tetracycline, Chlortetracycline, Oxytetracycline,Demeclocycline, Doxycycline, Lymecycline, Meclocycline, Methacycline,Minocycline, Rolitetracycline, Minocycline, Tigecycline, Chlorocycline,Glycylcyclines, Aminomethylcyclines, TP434, and PTK796, (also known asBAY 73-7388 and MK2764). The structure of TP434 is provided below:

In one embodiment, the tetracycline antibiotic is selected from thegroup consisting of tetracycline, oxytetracycline, doxycycline,chlorocycline, minocycline, glycylcyclines and aminomethylcyclines. Inone embodiment, the tetracycline is a glycylcycline. In one embodiment,the glycylcycline is tigecycline. In one embodiment, the tetracycline isan aminomethylcycline. In one embodiment, the aminomethylcycline isPTK796, also known as BAY 73-7388 and MK2764. In another embodiment, thetetracycline is selected from the group consisting of tetracycline,minocycline, tigecycline and PTK796. In one embodiment, the tetracyclineantibiotic is tetracycline. In one embodiment, of the invention, thetetracycline is minocycline. In one embodiment, of the invention, thetetracycline is tigecycline. In yet another embodiment, of theinvention, the tetracycline is PTK796. Some embodiments include a saltof a tetracycline antibiotic.

In some embodiments, the tetracycline antibiotic is a7-dimethylamino-tetracycline. 7-dimethylamino-tetracyclines contain anadditional dimethylamino substituent at the 7-position on the four-ringcore. The 7-position is indicated on following numbered structure ofminocycline:

Examples of 7-dimethylamino-tetracyclines include minocycline, aglycylcycline (e.g., tigecycline) and PTK796. Example structures of suchcompounds include:

As used herein, “glycylcyclines” are 7-dimethylamino-tetracyclineshaving an N-alkylglycylamido side chain at position 9 of the four-ringcore.

In some embodiments, the 7-dimethylamino-tetracycline antibiotic has thestructure:

or tautomers thereof, wherein:

-   -   R¹ is selected from H, —(CH₂)_(n)NHC(O)(CH₂)_(n)R¹⁰, and        —(CH₂)_(n)R¹⁰, where each n is independently an integer from 0        to 3, and    -   R¹⁰ is selected from —NH—C₁₋₈alkyl, —NH—C₁₋₈cycloalkyl, and a        saturated 4-to-7-membered heterocycle containing one nitrogen        atom, wherein if the connecting atom of R¹⁰ is carbon, the        nitrogen atom is optionally substituted by C₁-C₄alkyl;    -   Y is CR² or N; and    -   R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are each independently        selected from H, —OH, halogen, and C₁₋₄ alkyl; or    -   optionally R¹ and R² together form a 6-membered aryl or        heteroaryl ring, optionally substituted by one or two groups        independently selected from H, R¹, —OH, halogen, and C₁₋₄ alkyl.

In some embodiments, each of R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ arehydrogen.

As used herein, “alkyl” refers to a straight- or branched-chain moietycontaining only carbon and hydrogen. Alkyls may have any degree ofsaturation. Examples include methyl, ethyl, propyl, isopropyl, butyl,isobutyl, and tertbutyl.

As used herein, “cycloalkyl” refers to a ring or ring system comprisingonly carbon in the ring backbone. Cycloalkyls may include one or morefused or bridged rings. Cycloalkyls may have any degree of saturationprovided that at least one ring is not aromatic. Examples includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexenyl.

As used herein, “heterocycle” refers to a ring or ring system comprisingat least one heteroatom in the ring backbone. Heterocycles may includeone or more fused or bridged rings. Heterocycles may have any degree ofsaturation provided that at least one ring is not aromatic. Examplesinclude pyrrolidine, piperidine, piperazine, and morpholino.

As used herein, “aryl” refers to an aromatic ring or ring systemcomprising only carbon in the ring backbone. Aryls may include one ormore fused rings. Examples include phenyl and naphthyl.

As used herein, “heteroaryl” refers to an aromatic ring or ring systemcomprising at least one heteroatom in the ring backbone. Heteroaryls mayinclude one or more fused rings. Examples include imidazole, oxazole,pyridine, and quinoline.

Some compositions include at least one multivalent cation. Multivalentcations include bivalent and trivalent cations, e.g., metal cations. Themetal cations include common multivalent metal cations. In someembodiments, the metal cations include iron, copper, zinc, manganese,nickel, cobalt, aluminum, calcium, magnesium and gallium.

Some compositions include a salt that comprises the cation. In oneembodiment, the salts are inorganic metal salts and can includeanhydrous, hydrated and solvated forms of the salts. In anotherembodiment, the salts are organic metal salts and include but are notlimited to the anhydrous, hydrated and solvated forms of the salt. Inone embodiment, the anion in the inorganic metal salts can includechloride, bromide, oxide, and sulfate salts. In one embodiment, theorganic metal salts are those where the anion of the salt is selectedfrom the GRAS (generally regarded as safe) list such as but not limitedto acetate, citrate, gluconate, and malate salts. Suitable anions mayalso be found in see Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa. In some embodiments, a composition caninclude more than one type of metal cation. In some such embodiments,the anions for each metal salt can be the same. In another embodiment,the anions for each metal salt are different. In another embodiment, themetal cation is included in the compositions provided herein asdifferent salts of the same cation. In one embodiment the metal saltsare all inorganic. In another embodiment, the metal salts are allorganic. In yet another embodiment, the metal salts are a combination oforganic and inorganic salts.

Examples of inorganic metal salts that may be included in thecompositions provided herein include magnesium chloride (including thehexahydrate), magnesium bromide, magnesium sulfate (including theheptahydrate), magnesium oxide, calcium chloride, calcium bromide,calcium sulfate, zinc chloride, and gallium chloride. Examples ofinorganic metal salts that may be included in the compositions providedherein include magnesium malate, magnesium gluconate, magnesium citrate,magnesium acetate (including the trihydrate), calcium gluconate, calciumcitrate, zinc gluconate, zinc acetate, and zinc citrate. The saltsdescribed herein include both their anhydrous and hydrated forms.

Some compositions provided herein include a tetracycline and divalent ortrivalent cation, e.g., metal cation at particular molar ratios ofdivalent or trivalent cation to tetracycline. For example, someembodiments include compositions comprising a tetracycline and adivalent or trivalent cation, wherein the molar ratio of said divalentor trivalent cation to said tetracycline is greater than about 1:1. Insome such embodiments, the molar ratio of the divalent or trivalentcation to the tetracycline is greater than about 2:1, greater than about3:1, greater than about 4:1, greater than about 5:1, greater than about6:1, greater than about 7:1, greater than about 8:1, greater than about9:1, greater than about 10:1, greater than about 11:1, greater thanabout 12:1, greater than about 13:1, greater than about 14:1, greaterthan about 15:1, greater than about 16:1, greater than about 17:1,greater than about 18:1, greater than about 19:1, greater than about20:1, greater than about 21:1, greater than about 22:1, greater thanabout 23:1, greater than about 24:1, greater than about 25:1, greaterthan about 26:1, greater than about 27:1, greater than about 28:1,greater than about 29:1, and greater than about 30:1. In someembodiments, the molar ratio is greater than about 35:1, greater thanabout 40:1, greater than about 45:1, and greater than about 50:1,

In some such embodiments, the molar ratio of the divalent or trivalentcation to the tetracycline is between about 1:1 to about 30:1, betweenabout 5:1 to about 30:1, between about 10:1 to about 30:1, and betweenabout 20:1 to about 30:1. In some such embodiments, the molar ratio ofthe divalent or trivalent cation to the tetracycline is between about1:1 to about 50:1, between about 5:1 to about 50:1, between about 10:1to about 50:1, and between about 20:1 to about 50:1.

In some embodiments, the relative amounts of metal cation present in thecompositions of the invention are those amounts which are in excess ofthe 1:1 metal cation: a tetracycline stoichiometry for each metalcation. In one embodiment of the invention, the metal cation to atetracycline molar ratio ranges from 5:1 to 100:1. In another embodimentof the invention, the metal cation to a tetracycline molar ratio rangesfrom 5:1 to 50:1. In yet another embodiment of the invention, the metalcation to a tetracycline molar ratio ranges from 5:1 to 30:1. In oneembodiment of the invention, the metal cation to a tetracycline molarratio ranges from 5:1 to 10:1. In one embodiment of the invention, themetal cation to a tetracycline molar ratio ranges from 10:1 to 20:1. Inone embodiment of the invention, the metal cation to a tetracyclinemolar ratio ranges from 10:1 to 15:1. In one embodiment of theinvention, the metal cation to a tetracycline molar ratio is 5:1. In oneembodiment of the invention, the metal cation to a tetracycline molarratio is 10:1. In one embodiment of the invention, the metal cation to atetracycline molar ratio is 12:1. In one embodiment of the invention,the metal cation to a tetracycline molar ratio is 15:1. In oneembodiment of the invention, the metal cation to a tetracycline molarratio is 20:1. In one embodiment of the invention, the metal cation to atetracycline molar ratio is 30:1.

Some compositions include carbohydrates in addition to a divalent ortrivalent cation. Suitable carbohydrates are those carbohydrates capableof reducing degradation of the tetracycline in at least one solid formprepared in at least one pH environment when compared to a solid form ofa tetracycline prepared at the same pH environment lacking suitablecarbohydrates. In one embodiment, the pH environment ranges from 3.0 toabout 7.0, such as pHs ranging from about 4.0 to about 6.5, from about5.0 to about 6.5, and from about 5.5 to about 6.5. In one embodiment, atleast one solid form is chosen from powders and lyophilized cakes of atetracycline. In another embodiment of the invention, carbohydrates arethose carbohydrates capable of reducing degradation of the tetracyclinein solution prepared in at least one pH environment when compared to asolution of a tetracycline prepared at the same pH environment lackingsuitable carbohydrates. In one embodiment, the pH environment rangesfrom 3.0 to about 7.0, such as pHs ranging from about 4.0 to about 6.5,from about 5.0 to about 6.5, and from about 5.5 to about 6.5.

Suitable carbohydrates include mono and disaccharides e.g. an aldosemonosaccharide or a disaccharide. Examples of suitable carbohydratesinclude but are not limited to the anhydrous, hydrated and solvatedforms of compounds such as trehalose, lactose, mannose, sucrose andglucose. In one embodiment of the invention, the carbohydrate is adisaccharide. In another embodiment of the invention, the disaccharideis trehalose, lactose or sucrose. In yet another embodiment of theinvention, the carbohydrate is lactose, including its different formssuch as anhydrous lactose, lactose monohydrate or any other hydrated orsolvated form of lactose. In one embodiment of the invention, thecarbohydrate is trehalose, including its different forms such asanhydrous trehalose, trehalose dihydrate or any other hydrated orsolvated form of trehalose.

In one embodiment of the invention, the suitable carbohydrate used islactose monohydrate and the molar ratio of tigecycline to lactosemonohydrate in the lyophilized powder or cake is between 1:0.2 to about1:5. In another embodiment of the invention, the tigecycline to lactosemonohydrate molar ratio is between 1:1.6 to about 1:3.3.

Some compositions include an antioxidant. Antioxidants can be used toprevent or reduce the oxidation of tetracyclines either in solution orin the solid state. Examples of antioxidants include ascorbic acid,citric acid, trehalose, butylated hydroxyl toluene (BHT), butylatedhydroxyl anisole (BHA), sodium metabisulfite, d,l-α-tocopherol, andgentisic acid.

It will be appreciated that the compositions provided herein can includeaerosols, liquids, and solids. Solids can include, for example,lyophilized compositions, such as powders, cakes, or the like. Suchsolids may be water soluble so that they may be used to prepare aqueoussolutions. Liquids can include solutions or suspensions, which may beprepared from solid compositions. Liquids include solutions that may beprepared prior to manufacturing procedures such as lyophilization. Inone embodiment, the solution may be stored for several hours prior tolyophilization in order to provide greater manufacturing flexibility.Liquids also include solutions that are prepared by reconstitution foruse in administration to a patient. Some compositions include solutionsmade from the lyophilized powder or cake by, for example, reconstitutionwith saline or other pharmaceutically acceptable diluents.Pharmaceutically acceptable diluents are those listed by USP such as butnot limited to water for injection, saline solution, lactated Ringer'ssolution for injection or dextrose solution. Some compositions includesolutions resulting from diluting those reconstituted solutions withpharmaceutically acceptable diluents for use in intravenous bags.

In some embodiments, the pH of a liquid composition provided herein,such as an aqueous solution, is between about pH 2.0 to about pH 8.0,between about pH 2.5 to about pH 7.5. In some embodiments, the pH of thecomposition is between about pH 3.0 to about pH 7.0, between about pH3.5 to about pH 6.5, between about pH 4.0 to about pH 6.5, between aboutpH 4.0 to about pH 6.0, between about pH 4.5 to about pH 6.0, betweenabout pH 4.5 to about pH 5.5, between about pH 5.0 to about pH 5.5,between about pH 5.5 to about pH 6.5, between about pH 3.5 to about pH4.5. In some embodiments, the pH of the solution is less than pH 7, lessthan pH 6, less than pH 5, less than pH 4, less than pH 3, and less thanpH 2. In some embodiments the pH of the solution is greater than pH 2and less than pH 7, greater than pH 4 and less than pH 7, greater thanpH 4 and less than pH 6, and greater than pH 4 and less than pH 5.

In some embodiments, liquid compositions, such as an aqueous solution,can have an osmolality from about 300 mOsmol/kg to about 500 mOsmol/kg,from about 325 mOsmol/kg to about 450 mOsmol/kg, from about 350mOsmol/kg to about 425 mOsmol/kg, or from about 350 mOsmol/kg to about400 mOsmol/kg. In some embodiments, the osmolality of the formulation isgreater than about 300 mOsmol/kg, about 325 mOsmol/kg, about 350mOsmol/kg, about 375 mOsmol/kg, about 400 mOsmol/kg, about 425mOsmol/kg, about 450 mOsmol/kg, about 475 mOsmol/kg, or about 500mOsmol/kg. In some embodiments, liquid compositions can have anosmolality from about 200 mOsmol/kg to about 1250 mOsmol/kg. In anotherembodiment, the osmolality is between about 250 mOsmol/kg and about 1050mOsmol/kg. In another embodiment, the osmolality is between about 250mOsmol/kg and about 750 mOsmol/kg. In another embodiment, the osmolalityis between about 350 mOsmol/kg and about 500 mOsmol/kg. In someembodiments, the osmolality of the solution is less than 500 mOsmol/kg,450 mOsmol/kg, 400 mOsmol/kg, 350 mOsmol/kg, or 300 mOsmol/kg.

Some embodiments include an aqueous solution comprising a tetracyclinehaving a concentration of at least 1 mg/ml, 5 mg/ml, 10 mg/ml, 15 mg/ml,20 mg/ml, 25 mg/ml, 30 mg/ml, 35 mg/ml, 40 mg/ml, 45 mg/ml, or 50 mg/ml.

Some embodiments include an aqueous solution comprising a buffer, suchas an acetate buffer (e.g., provided as sodium acetate), wherein theacetate has a concentration of at least 0.01 M, 0.02 M, 0.03 M, 0.04 M,0.05 M, 0.1 M, 0.15 M, 0.20 M, 0.25 M, 0.30 M, 0.35 M, 0.40 M, 0.45 M,0.50 M, 0.55 M, 0.60 M, 0.65 M, 0.70 M, 0.75 M, 0.80 M, 0.85 M, 0.90 M,or 0.95 M.

Some embodiments include an aqueous solution comprising a saltcomprising divalent or trivalent cation, such as a magnesium salt (e.g.,magnesium chloride or magnesium sulfate), having a concentration of atleast 0.01 M, 0.02 M, 0.03 M, 0.04 M, 0.05 M, 0.1 M, 0.15 M, 0.20 M,0.25 M, 0.30 M, 0.35 M, 0.40 M, 0.45 M, 0.50 M, 0.55 M, 0.60 M, 0.65 M,0.70 M, 0.75 M, 0.80 M, 0.85 M, 0.90 M, or 0.95 M.

In one embodiment, liquid compositions, such as aqueous solutions, havea permeant ion concentration from about 30 mM to about 300 mM. Inanother embodiment, the permeant ion concentration is between 50 mM and200 mM. In another embodiment, the permeant ion is selected from thelist consisting of chloride and bromide. In another embodiment thepermeant ion is chloride. In another embodiment, the permeant ion isbromide.

In some embodiments, aqueous solution compositions comprise a buffer.For example, in some embodiments, the solution comprises acetate. Insome embodiments, aqueous solution compositions comprise a base such asNaOH. In some embodiments, aqueous solution compositions comprise anacid such as HCl.

It is contemplated that in some embodiments, reconstituted solutions maybe stored in a reconstituted state at room temperature prior to furtherdilution for injection or topical administration. In some embodiments,storage times at room temperature after reconstitution are much longerthan current compositions. In some embodiments, admixing can occur, forexample, in an intravenous bag. To prepare an admixture, sufficientreconstituted solution is mixed in an intravenous bag containing apharmaceutically acceptable diluent such as saline or dextrose solutionsuch as 5DW.

The concentration of admixtures may easily be determined by those ofordinary skill in the art. The time available for admixture ofreconstituted solutions from the compositions may be much longer thanthose of previously described formulations. Storage times of theadmixtures at room temperature may also be much longer than those of theexisting compositions. Once admixed, the tetracycline solution is readyfor administration by or to the patient. The admixture may beadministered alone or together with another pharmaceutical agent orcomposition.

In some embodiments, the composition does not comprise apharmaceutically acceptable oil. In some embodiments, an oil can referto a hydrocarbon compound that is liquid at room temperature andinsoluble in water. Examples of pharmaceutically acceptable oils includepolyoxyethylene hydrogenated castor oils such as PEG-40 hydrogenatedcastor oil and PEG-50 hydrogenated castor oil. More examples ofpharmaceutically acceptable oils include olive oil, sesame oil, soybeanoil, safflower oil, cottonseed oil, corn oil, sunflower oil, arachisoil, coconut oil, an omega-3 polyunsaturated oil, and an omega-3 marinetriglyceride.

In some embodiments, the composition does not comprise apyridine-containing compound. In one embodiment, the pyridine-containingcompound is nicotinamide.

Although some embodiments include gluconate (e.g., as the gluconate saltof a divalent or trivalent metal cation), other embodiments includecompositions that do not comprise gluconate.

In some embodiments, the composition does not comprise a non-aqueoustetracycline-solubilizing co-solvent. Such solubilizing co-solvents caninclude the oil, pyridine-containing compound, and gluconate describedabove.

Although some embodiments include an antioxidant, other embodimentsinclude compositions that do not comprise an antioxidant (e.g., sodiumor magnesium formaldehyde sulfoxylate; sodium sulfite, metabisulfite orbisulfite; sodium sulfide; alpha-monothioglycerol (also referred to asthioglycerol); and thiosorbitol).

Other various embodiments include compositions that do not include oneor more of an alcohol (e.g., a polyhydric alcohol, such as, propyleneglycol, ethylene glycol), glycerol, polyethylene glycol, apyrrolidone-containing compound, a water-miscible local anaesthetic(e.g., procaine, tetracaine), urea, lactose, or a dehydrating agent(e.g., ethyl acetate, acetic anhydride, absolute ethanol, ethyl acetate,acetic anhydride, and mixtures thereof).

Some embodiments include compositions comprising a7-dimethylamino-tetracycline and a cation. In some such embodiments the7-dimethylamino-tetracycline is minocycline. In some embodiments, theminocycline is minocycline HCl. In some embodiments the cation comprisesMg²⁺. In some embodiments, the compositions include a salt selected fromMgCl₂ (e.g., MgCl₂.6H₂O), MgSO₄ (e.g. MgSO₄.7H₂O) and magnesium acetate(e.g., Mg(CH₃COO)₂.3H₂O). In some embodiments, the molar ratio ofdivalent or trivalent cation to minocycline is greater than 1:1, 2:1,3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, themolar ratio of divalent or trivalent cation to minocycline is greaterthan 10:1, 20:1, 30:1, 40:1, or 50:1. Some embodiments include a buffer.In some such embodiments, the buffer includes NaOH, or sodium acetate(e.g., NaCH₃COO.3H₂O).

Some compositions comprise minocycline and MgCl₂.6H₂O with a Mg tominocycline molar ratio of about 5:1 in a base comprising NaOH. Somesuch embodiments are suitable for intravenous use.

Some compositions comprise minocycline and MgSO₄.7H₂O with a Mg tominocycline molar ratio of about 5:1 in a buffer comprisingNaCH₃COO.3H₂O with a pH in the range 4.5-5.5 and an osmolality in therange of about 275-375 mOsm/kg. Some such compositions can be preparedas an aqueous solution and lyophilized. As will be understood by askilled artisan, the pH and osmolality of a reconstituted solution canhave a pH in the range 4.5-5.5 and an osmolality in the range of about275-375 mOsm/kg. Some such embodiments are suitable for intravenous use.

Some embodiments comprise minocycline and Mg(CH₃COO)₂.3H₂O with a Mg tominocycline molar ratio of about 5:1 with no buffer added. Some suchembodiments are suitable for intravenous use.

Some embodiments include minocycline and MgSO₄.7H₂O with a Mg tominocycline molar ratio of about 5:1 in a base comprising NaOH with a pHin the range 5.5-6.5. Some such compositions can be prepared as anaqueous solution and lyophilized. As will be understood by a skilledartisan, the pH of a reconstituted solution can have a pH in the range5.5-6.5. Some such embodiments are suitable for intravenous use.

Some embodiments comprise tigecycline and MgSO₄.7H₂O with a Mg tominocycline molar ratio of about 5:1 in a buffer comprising NaOH with apH in the range 5.5-6.5. Some embodiments comprise tigecycline andMgSO₄.7H₂O with a Mg to minocycline molar ratio of about 12:1 in a basecomprising NaOH with a pH in the range 5.5-6.5. Some such compositionscan be prepared as an aqueous solution and lyophilized. As will beunderstood by a skilled artisan, the pH of a reconstituted solution canhave a pH in the range 5.5-6.5. Some such embodiments are suitable forintravenous use.

Some embodiments comprise tigecycline and MgCl₂.6H₂O with a Mg tominocycline molar ratio of about 5:1 in a buffer comprising NaOH with apH in the range 5.5-6.5. Some embodiments comprise tigecycline andMgCl₂.6H₂O with a Mg to minocycline molar ratio of about 12:1 in a basecomprising NaOH with a pH in the range 5.5-6.5. Some such compositionscan be prepared as an aqueous solution and lyophilized. As will beunderstood by a skilled artisan, the pH of a reconstituted solution canhave a pH in the range 5.5-6.5. Some such embodiments are suitable forintravenous use.

Some embodiments comprise tigecycline and MgSO₄.7H₂O with a Mg tominocycline molar ratio of about 5:1 in a buffer comprising NaOH with apH in the range 6.0-7.0. Some embodiments comprise tigecycline andMgSO₄.7H₂O with a Mg to minocycline molar ratio of about 12:1 in a basecomprising NaOH with a pH in the range 6.0-7.0. Some such compositionscan be prepared as an aqueous solution and lyophilized. As will beunderstood by a skilled artisan, the pH of a reconstituted solution canhave a pH in the range 6.0-7.0. Some such embodiments are suitable fortopical use. Some such compositions comprise tigecycline with greaterthan 90%, 95%, or 98% stability for at least 30 days. Some embodimentsinclude compositions comprising an additional constituent such asbenzalkonium chloride, a steroid such as hydrocortisone, dexamethasone,thonzonium bromide, tyloxapol, an antiseptic agent such as boric acid, apreservative such as benzalkonium chloride.

Some embodiments comprise tigecycline and CaCl₂.6H₂O with aCa:minocycline:molar ratio of about 5:1 in a base comprising NaOH with apH in the range 6.0-7.0. Some embodiments comprise tigecycline andCaCl₂.6H₂O with a Ca to tigecycline molar ratio of about 12:1 in a basecomprising NaOH with a pH in the range 6.0-7.0. Some such compositionscan be prepared as an aqueous solution and lyophilized. As will beunderstood by a skilled artisan, the pH of a reconstituted solution canhave a pH in the range 6.0-7.0. Some such embodiments are suitable fortopical use. Some such compositions comprise tigecycline with greaterthan 90%, 95%, 98% stability for at least 30 days. Some embodimentsinclude compositions comprising an additional constituent such asbenzalkonium chloride, a steroid such as hydrocortisone, dexamethasone,thonzonium bromide, tyloxapol, an antiseptic agent such as boric acid, apreservative such as benzalkonium chloride.

Some embodiments include pharmaceutical compositions comprising anaqueous solution of minocycline and a divalent or trivalent cation,wherein the molar ratio of divalent or trivalent cation to minocyclineis greater than 2:1. In some embodiments, the molar ratio of divalent ortrivalent cation to minocycline is greater than about 3:1, greater thanabout 5:1, greater than about 8:1, greater than about 10:1. In someembodiments, the divalent or trivalent cation is selected from iron,copper, zinc, manganese, nickel, cobalt, aluminum, calcium, magnesiumand gallium. In particular embodiments, the divalent or trivalent cationis selected from magnesium, calcium, and zinc. In some embodiments, thesolution comprises magnesium sulfate and/or magnesium oxide. Inparticular embodiments, the composition is suitable for intravenousadministration.

More embodiments include a pharmaceutical composition comprising anaqueous solution of an 7-dimethylamino-tetracycline antibiotic and adivalent or trivalent cation, wherein the molar ratio of divalent ortrivalent cation to tetracycline antibiotic is greater than 3:1 andwherein the solution does not comprise an oil, gluconate, or apyridine-containing compound, has a pH greater than 2 and less than 7,and is suitable for intravenous administration. In some embodiments, the7-dimethylamino-tetracycline is selected from minocycline, PTK796, andglycylcyclines (e.g. tigecycline).

Some embodiments include a water-soluble solid composition, comprisingminocycline or a salt thereof and a salt that comprises a divalent ortrivalent cation. In some embodiments, the molar ratio of divalent ortrivalent cation to minocycline is greater than about 1:1, greater thanabout 2:1, greater than about 3:1, greater than about 5:1, greater thanabout 8:1, greater than about 10:1. In some embodiments, the salt isselected from magnesium chloride, magnesium bromide, magnesium sulfate,calcium chloride, calcium bromide, calcium sulfate, zinc chloride,gallium chloride, magnesium malate, magnesium gluconate, magnesiumcitrate, calcium gluconate, calcium citrate, zinc gluconate, zincacetate, and zinc citrate. In preferred embodiments, the salt ismagnesium sulfate. In some embodiments, the composition comprises sodiumacetate. In certain embodiments, the composition does not comprise anantioxidant, a pyridine-containing compound (e.g., nicotinamide), orgluconate.

More embodiments include water-soluble solid compositions comprising a7-dimethylamino-tetracycline antibiotic and a salt comprising a divalentor trivalent cation, wherein the molar ratio of divalent or trivalentcation to tetracycline antibiotic is greater than 3:1 and wherein thecomposition does not comprise gluconate or a pyridine-containingcompound. In some embodiments, the 7-dimethylamino-tetracycline isselected from minocycline, glycylcyclines (e.g. tigecycline) and PTK796.

In some embodiments, the water-soluble compositions described above arein the form of a lyophile.

Methods of Preparation

Some embodiments of the present invention include methods for preparingthe compositions described herein. Some such methods include combining atetracycline antibiotic and a divalent or trivalent cation. Some methodsfurther comprise modifying the pH of the compositions. In some methods,modifying the pH comprises adjusting the pH with a pH modifying agent.Examples of pH modifying agents include hydrochloric acid, gentisicacid, lactic acid, citric acid, acetic acid, phosphoric acid, sodiumhydroxide, sodium bicarbonate and sodium carbonate. In some embodiments,the pH-modifying agent includes any pharmaceutically acceptable acid,base or buffer capable of adjusting the pH of a tetracyclineantibiotic/metal cation solution to between about 3.0 to about 7.0,about 4.0 to about 5.0, about 5.0 to 6.0, about 5.5 to 6.5, about 6.0 to6.5 or about 4.2 to 4.8. In some embodiments, the acid, base or bufferis used to adjust the pH of a tetracycline antibiotic/metal cationsolution to a pH less than 7, 6, 5, or 4. In some embodiments, the acid,base or buffer is used to adjust the pH of a tetracyclineantibiotic/metal cation solution to a pH greater than 2 and less than 7,greater than 4 and less than 7, greater than 4 and less than 6, andgreater than 4 and less than 5. Examples of such acids include but arenot limited to hydrochloric acid, including 1.0 N HCl, gentisic acid,lactic acid, citric acid, acetic acid and phosphoric acid. Examples ofsuitable buffers include as components succinates and acetate. Examplesof such bases include but are not limited to aqueous solutions of sodiumhydroxide, including 1.0 N NaOH solution, sodium bicarbonate and sodiumcarbonate.

Compositions of the invention may be prepared via a number of acceptablemethods. For example, the metal salts are dissolved in water and thetetracycline antibiotic is added to this solution. Alternatively, theantibiotic is dissolved first and the metal salt is added to thesolution. The pH of the solution is then adjusted with an acid, a baseor buffer. Other optional agents such as an antioxidant or carbohydrateare then dissolved in the solution. The final solution may be then beused directly in therapy or lyophilized to dryness to form a lyophilizedpowder or cake for later reconstitution.

In another example, a tetracycline antibiotic may be dry blended withthe metal salts and other optional ingredients, and the residual mixturedissolved in water. After the pH of the solution is adjusted, thesolution may then be used in therapy or lyophilized to dryness to form apowder or cake.

Lyophilization of solutions described herein may be accomplished by anypharmaceutically acceptable means. Once lyophilized, the compositions ofthe invention may be stored under an inert gas, such as nitrogen, tofurther slow the degradation process.

The tetracycline antibiotic used in the various preparation techniquesmay be any solid-state form of the tetracycline that is sufficientlysoluble in water. Such solid-state forms include crystallinetetracycline polymorphs, amorphous forms and salts.

One embodiment for preparing a minocycline-containing pharmaceuticalcomposition includes dissolving minocycline and a salt that comprises adivalent of trivalent cation in water to form a solution and adjustingthe pH of the solution to be less than about 7, less than about 6, lessthan about 5, less than about 4, or less than about 3. In someembodiments, the pH of the solution is adjusted to be greater than about2 and less than about 7, greater than about 4 and less than about 7, orgreater than about 4 and less than about 6. In some embodiments,adjusting the pH comprises adding a base, e.g., NaOH. In someembodiments, adjusting the pH comprises forming a buffer. In someembodiments, forming the buffer comprises adding sodium acetate.

More embodiments for methods of preparing a minocycline-containingpharmaceutical composition includes dissolving minocycline in a solutioncomprising a divalent or trivalent cation; and adjusting the pH of thesolution to be less than 7.

In some embodiments, a solution of a 7-dimethylamino-tetracycline can beprepared by adding a 7-dimethylamino-tetracycline, an aqueous solutionof divalent or trivalent salt to provide a certain divalent or trivalentsalt to 7-dimethylamino-tetracycline molar ratio. The pH of the solutioncan be adjusted to a certain pH with a buffer, acid, or a base. Theosmolality of the solution can be adjusted to a certain osmolality. Thesolution can be lyophilized. The lyophilized solution can bereconstituted with a diluent such as water.

In some embodiments, a solution of a 7-dimethylamino-tetracycline can beprepared by adding a 7-dimethylamino-tetracycline to an acid, such asHCl. The solution can be lyophilized. The lyophilized solution can bereconstituted with a diluent comprising a divalent or trivalent salt toprovide a certain divalent or trivalent salt to7-dimethylamino-tetracycline molar ratio. The diluent can furthercomprise an acid, base, or buffer, such as sodium acetate, to provide asolution of a certain pH.

In some embodiments, minocycline can be in a buffer comprising MgSO₄ atpH 5. The solution can be lyophilized. The lyophilisate can bereconstituted in an aqueous diluent. In some embodiments, minocyclinecan be solubilized in an aqueous solution comprising HCl, MgSO4 andsodium acetate. The solution can be lyophilized. In some embodiments,minocycline can be solubilized in an aqueous solution comprising HCl.The solution can be lyophilized. The lyophilisate can be reconstitutedin an aqueous solution. In some embodiments, the reconstituting solutioncan lack Mg.

Kits

Some embodiments of the present invention include kits comprising acomposition described herein. Some kits include a single use containercomprising a composition described herein. Single use containers includeampules, vials, and the like. The single-use container can comprise alyophilized formulation of a composition described herein. Some kitsinclude a diluent for reconstituting the lyophilized formulations of acomposition or pharmaceutical composition described herein.

In some embodiments, the compositions of the invention may be preparedfor single-dosage use. In this embodiment, the solutions of theinvention are lyophilized in individual vials such as 20-mL vials. Uponlyophilization, the vials are stoppered with any acceptable stopper. Thestoppered vials are then shipped for use. When needed, the vials can bereconstituted by adding sufficient diluents to achieve the desiredconcentration of tetracycline. The concentration of reconstitutedsolutions may be easily determined by those of ordinary skill in theart. Any pharmaceutically acceptable diluent may be used. Examples ofsuch diluents include but are not limited to water, 0.9% saline,Lactated Ringer's injection solution and dextrose solutions including 5%dextrose (5DW).

In some embodiments, the diluent does not comprise a pharmaceuticallyacceptable oil (e.g., polyoxyethylene hydrogenated castor oils), apyridine-containing compound (e.g., nicotinamide), gluconate, anantioxidant, an alcohol (e.g., a polyhydric alcohol, such as, propyleneglycol, ethylene glycol), glycerol, polyethylene glycol, apyrrolidone-containing compound, a water-miscible local anaesthetic(e.g., procaine, tetracaine), urea, lactose, or a dehydrating agent(e.g., ethyl acetate, acetic anhydride, absolute ethanol, ethyl acetate,acetic anhydride, and mixtures thereof). In some embodiments, thediluent does not comprise a tetracycline-solubilizing cosolvent.

In some embodiments, the diluent contains the divalent or trivalentcation. For example, some embodiments include kits that comprise a firstcontainer comprising a diluent that comprises an aqueous solution of adivalent or trivalent cation; and a second container comprising a solidcomposition soluble in the diluent, wherein the solid compositioncomprises minocycline in an amount such that the molar ratio of thedivalent or trivalent cation to minocycline is greater than about 2:1.In some embodiments, the diluent comprises an acid, e.g., HCl. In someembodiments, the diluent comprises a buffer. In some embodiments, thebuffer is sodium acetate.

More embodiments include kits comprising a first container comprising adiluent that comprises an aqueous solution of a divalent or trivalentcation; and a second container comprising a solid composition soluble inthe diluent, wherein the solid composition comprises a tetracyclineantibiotic in an amount such that the molar ratio of the divalent ortrivalent cation to tetracycline antibiotic is greater than 3:1.

More embodiments include single use vials comprising any compositionwherein the vial comprises an amount of a tetracycline of at least 100μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1000μg. In some embodiments, the vial comprises an amount of a tetracyclineof at least 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg,45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, and 130 mg. In someembodiments, the vial comprises an amount of a tetracycline of at least100 mg, 200 mg, 300 mg, 400 mg, and 500 mg. In some embodiments, thevial comprises about 100 mg of a tetracycline. In some embodiments, thetetracycline is minocycline. In some embodiments, the tetracycline istigecycline. In some such embodiments, a vial can comprise greater than30 mg and less than 100 mg tigecycline.

Methods of Treatment

Some embodiments include methods of treating or preventing a bacterialinfection in a subject by administering a composition described herein.“Treating,” as used herein, refers to administering a pharmaceuticalcomposition for therapeutic purposes to a patient suffering from abacterial infection. “Preventing,” as used herein, refers to treating apatient who is not yet infected, but who is susceptible to, or otherwiseat risk of, a particular infection, whereby the treatment reduces thelikelihood that the patient will develop an infection.

In some embodiments, the administration is via an intravenous route suchas by administering an aqueous solution described herein intravenously.

Some such methods include administering an aqueous solution ofminocycline and a divalent or trivalent cation to a subject via anintravenous route. Such solutions are described herein.

Some embodiments include administering an aqueous solution of a7-dimethylamino-tetracycline antibiotic and a divalent or trivalentcation to a subject via an intravenous route, wherein the molar ratio ofdivalent or trivalent cation to tetracycline antibiotic is greater thanabout 3:1 and wherein the solution does not comprise gluconate or apyridine-containing compound and has a pH greater than 2 and less than7.

In some embodiments of intravenous administration, the compositionsdescribed herein permit use of lower volumes and faster infusion timesdue to increased concentrations of tetracycline antibiotic and reducedinjection site phlebitis as compared to currently available intravenousformulations. In some embodiments, the total volume administered is lessthan 50 ml, less than 60 ml, less than 70 ml, less than 80 ml, less than90 ml, less than 100 ml, less than 110 ml, less than 120 ml, less than130 ml, less than 140 ml, less than 150 ml, less than 200 ml, less than300 ml, less than 400 ml, less than 500 ml, or less than 1000 ml. Insome embodiments, about 100 ml is administered. In some embodiments, theentire volume to be administered is administered in less than 10minutes, less than 20 minutes, less than 30 minutes, less than 40minutes, less than 50 minutes, less than 60 minutes, less than 70minutes, less than 80 minutes, less than 90 minutes, less than 2 hours,less than 3 hours, or less than 4 hours. In some embodiments, the entirevolume is administered in 20-70 minutes. In some embodiments, the entirevolume is administered in 30-60 minutes.

Some embodiments include administering a composition described herein bya topical route. Examples of topical routes include skin, eye, ear,rectal, vaginal, urethral. Methods of such administration are well knownin the art and can include aqueous solution, spray, suppository, salve,or an ointment or the like. Accordingly, some embodiments includeadministering an aqueous solution of a 7-dimethylamino-tetracyclineantibiotic and a divalent or trivalent cation to a subject via a topicalroute. In some such embodiments, the molar ratio of divalent ortrivalent cation to tetracycline antibiotic is greater than about 3:1.In some embodiments, the solution does not comprise gluconate or apyridine-containing compound. In some embodiments, the solution has a pHgreater than 2 and less than 7.

Other embodiments include administering a composition described hereinby pulmonary inhalation. For example, compositions may be administeredby inhalation of an aerosol of the composition. The aerosol may beformed using dry particles of the composition or by nebulization of asolution of suspension of the composition. Any suitable aerosolizationdevice may be used, including dry-powder inhalers, metered-doseinhalers, and nebulizers.

The following examples illustrate various embodiments of the inventionand are not intended to limit the invention in any way.

EXAMPLES Example 1 Stability at 37° C. for Solutions of Tigecycline orTygacil® Containing Metal Cations

General procedures: Some of following examples include experiments inwhich the stabilities of various aqueous solutions of a tetracyclinewere analyzed. Some solutions included a carbohydrate and/or variousmolar amounts of metal salts.

The pH of the solutions were adjusted with hydrochloric acid or sodiumhydroxide solution. The solutions were incubated at room temperature(approximately 22° C.) or at 37° C. Incubation of solutions at 37° C.was used as a model for long-term storage of solutions.

The stabilities of various aqueous solutions of a tetracycline wereanalyzed using HPLC. HPLC analyses were conducted on an Agilent 1200:Column: Eclipse Plus C18 4.6×150 mm, 5 μm. Detection: UV at 248 nm. Flowrate: 1.2 mL/min. Tigecycline retention time=4.30 min. Gradient: SolventA=0.1% trifluoroacetic acid in acetonitrile. Solvent B=0.1%trifluroacetic acid in water. TABLE 1 shows the HPLC gradient used.

TABLE 1 Time (min) % Solvent A % Solvent B 0.0 5 95 9.5 50 50 10.0 5 9515.0 5 95

A 10 mg/mL Tigecycline aqueous solution was prepared and 300 μL aliquotsdispensed into polypropylene tubes. The volume of each tube was adjustedto 1 ml with various dilutions of 0.1 M MgCl₂, 0.1 M CaCl₂ or 0.1 MZnCl₂ to achieve the desired molar ratio of Tigecycline:metal cation.The tubes were incubated in the dark at 37° C. Samples of each solutionwere taken at various time points and analyzed by HPLC. The fraction ofremaining Tigecycline in each sample was determined.

A 10 mg/mL (17.08 mol/L) aqueous solution of Tygacil® (Lot D 90293, 53mg), a commercial Tigecycline formulation containing lactose, wasprepared, and 240 μL aliquots were dispensed into polypropylene tubes.The volume of each tube was adjusted to 1 ml with various dilutions of0.1 M MgCl₂, 0.1 M CaCl₂ or 0.1 M ZnCl₂ to achieve the desired molarratio of Tigecycline:metal cation. The tubes containing the solutionwere incubated in the dark at 37° C. Samples of each solution were takenat various time points and analyzed by HPLC. The fraction of remainingTigecycline in each sample was determined.

The percentages of Tigecycline remaining at Day 0, 1, 2, 5, and 7 forsolutions of Tigecycline at various molar ratios with MgCl₂, CaCl₂, orZnCl₂ are shown in TABLE 2, TABLE 3, and TABLE 4, respectively. Thepercentages of Tigecycline remaining at Day 0, 1, 2, 5, and 7 forsolutions of Tygacil® at various ratios with MgCl₂, CaCl₂, or ZnCl₂ areshown in TABLE 5, TABLE 6, and TABLE 7, respectively.

TABLE 2 MgCl₂:Tigecycline Molar ratio Day 0 Day 1 Day 2 Day 5 Day 710:1  99.42 98.93 97.68 92.31 85.95 5:1 99.45 98.85 97.30 88.64 81.412:1 99.50 98.57 96.85 84.95 73.95 1:1 99.64 98.64 96.70 82.54 67.870.5:1  99.60 98.45 96.52 79.39 62.20 0.2:1  99.56 98.44 95.91 72.8153.83 0.1:1  99.50 98.29 95.66 67.28 48.68 0:1 99.53 98.23 95.18 58.4240.90

TABLE 3 CaCl₂:Tigecycline Molar ratio Day 0 Day 1 Day 2 Day 5 Day 710:1  99.49 99.02 97.89 91.88 86.31 5:1 99.44 98.66 97.31 87.13 80.872:1 99.38 98.06 96.66 83.63 75.05 1:1 99.58 98.33 96.54 81.30 70.180.5:1  99.56 98.61 96.15 76.00 64.81 0.2:1  99.58 98.47 95.99 72.8457.19 0.1:1  99.56 98.32 95.66 67.89 49.75 0:1 99.49 98.17 94.98 59.1139.31

TABLE 4 ZnCl₂:Tigecycline Molar ratio Day 0 Day 1 Day 2 Day 5 Day 710:1  99.15 99.01 97.82 96.65 95.41 5:1 99.21 98.66 97.76 95.81 92.852:1 99.31 98.46 97.32 91.02 85.64 1:1 99.54 98.66 97.59 91.27 82.490.5:1  99.53 98.66 97.21 87.15 76.43 0.2:1  99.52 98.38 95.95 79.0866.83 0.1:1  99.50 98.39 96.11 78.80 64.93 0:1 99.46 98.37 95.02 56.3039.05

TABLE 5 MgCl₂:Tygacil ® Molar ratio Day 0 Day 1 Day 2 Day 5 Day 7  10:199.61 99.38 98.97 96.51 93.52  5:1 99.47 99.46 98.83 95.38 90.55  2:199.49 99.32 98.72 93.20 84.03  1:1 99.63 99.38 98.55 89.21 74.30 0.5:199.59 99.28 98.36 86.97 68.84 0.2:1 99.54 99.26 98.43 86.41 64.91 0.1:199.48 99.19 98.19 72.43 44.71

TABLE 6 CaCl₂:Tygacil ® Molar ratio Day 0 Day 1 Day 2 Day 5 Day 7 10:1 99.41 99.41 98.88 96.51 89.98 5:1 99.40 99.29 98.48 95.38 85.50 2:199.45 99.22 98.34 93.20 79.62 1:1 99.71 99.44 98.44 89.21 75.34 0.5:1 99.53 99.16 98.32 86.97 70.45 0.2:1  99.54 99.21 98.30 86.41 63.78 0:199.47 99.16 98.16 72.43 42.88

TABLE 7 ZnCl₂:Tygacil ® Molar ratio Day 0 Day 1 Day 2 Day 5 Day 7 10:1 99.41 99.45 98.90 97.89 95.78 5:1 99.44 99.27 98.68 96.87 94.30 2:199.39 99.25 98.74 96.09 92.22 1:1 99.56 99.50 98.98 95.60 90.67 0.5:1 99.48 99.25 98.78 93.73 86.02 0.2:1  99.52 99.35 98.43 89.34 77.79 0:199.50 99.27 98.12 69.85 42.15

While Tigecycline decomposed in all tubes over 7 days, the rate ofdecomposition was significantly lower in solutions containing highermolar ratios of metal cation. The rates of Tigecycline decomposition inthe presence of calcium or magnesium cations were similar; however, therate of Tigecycline decomposition in the presence of zinc wassignificantly lower. The presence of lactose in the Tygacil® formulationfurther decreased the rate of decomposition.

Example 2 Stability at Room Temperature for Solutions of Tigecycline orTygacil® Containing Metal Cations

A 10 mg/mL Tigecycline aqueous solution was prepared and 240 μL aliquotsdispensed into polypropylene tubes. The volume of each tube was adjustedto 1 ml with various dilutions of 0.1 M MgCl₂, 0.1 M CaCl₂ or 0.1 MZnCl₂ to achieve the desired molar ratio of Tigecycline:metal cation.The tubes were incubated in the dark at 37° C. Samples of each solutionwere taken at various time points and analyzed by HPLC. The fraction ofremaining Tigecycline in each sample was determined.

A 10 mg/mL aqueous solution of Tygacil® (Lot D 90293, 53 mg) wasprepared, and 240 μL aliquots were dispensed into polypropylene tubes.The volume of each tube was adjusted to 1 ml with various dilutions of0.1 M MgCl₂, 0.1 M CaCl₂ or 0.1 M ZnCl₂ to achieve the desired molarratio of Tigecycline:metal cation. The tubes were incubated in the darkat 37° C. Samples of each solution were taken at various time points andanalyzed by HPLC. The fraction of remaining Tigecycline in each samplewas determined.

The percentages of Tigecycline remaining at Day 0, 1, 2, 5, 7, 14, 21,28, and 36 for solutions of Tigecycline at various molar ratios withMgCl₂, CaCl₂, or ZnCl₂ are shown in TABLE 8, TABLE 9, and TABLE 10,respectively. The percentages of Tigecycline remaining at Day 0, 1, 2,5, 7, 14, 21, 28, and 36 for solutions of Tygacil® at various ratioswith MgCl₂, CaCl₂, or ZnCl₂ are shown in TABLE 11, TABLE 12, and TABLE13, respectively.

TABLE 8 MgCl₂:Tigecycline Molar ratio Day 0 Day 1 Day 2 Day 5 Day 7 Day14 Day 21 Day 28 Day 36 10:1  99.58 99.32 99.46 99.03 98.62 95.52 91.9085.33 76.89 5:1 99.45 99.32 99.41 98.74 98.16 94.04 87.10 76.71 62.602:1 99.51 99.27 99.43 98.46 96.97 89.87 76.29 58.07 40.67 1:1 99.6699.45 99.36 98.35 96.49 85.88 66.59 46.07 31.90 0.5:1   99.64 99.4099.35 97.76 96.16 81.98 59.70 39.79 28.16 0.2:1   99.56 99.37 99.2897.93 95.45 75.81 50.38 34.00 24.19 0:1 99.46 99.24 99.15 97.01 94.0861.98 38.99 24.55 16.33

TABLE 9 CaCl₂:Tigecycline Molar ratio Day 0 Day 1 Day 2 Day 5 Day 7 Day14 Day 21 Day 28 Day 36 10:1  99.58 99.34 99.41 99.05 98.59 95.45 92.0086.92 82.47 5:1 99.48 99.25 99.27 98.66 98.13 93.61 88.60 81.75 74.952:1 99.37 99.27 99.25 98.03 97.16 91.36 82.92 72.83 62.43 1:1 99.5799.38 99.30 98.53 96.92 89.14 78.35 65.46 53.22 0.5:1   99.59 99.3099.30 98.32 96.54 86.26 72.73 58.20 45.11 0.2:1   99.48 99.32 99.2797.94 95.75 80.39 61.83 45.47 26.69 0:1 99.44 99.29 99.17 96.76 93.7560.72 38.08 23.94 15.72

TABLE 10 ZnCl₂:Tigecycline Molar ratio Day 0 Day 1 Day 2 Day 5 Day 7 Day14 Day 21 Day 28 Day 36 10:1  99.24 98.99 99.49 99.30 99.19 97.49 97.6396.09 94.32 5:1 99.29 99.13 99.05 99.27 99.16 97.40 95.98 92.80 90.602:1 99.34 99.23 99.51 99.06 98.82 95.79 93.63 86.84 80.66 1:1 99.5399.39 99.47 99.03 98.48 94.61 88.48 79.03 69.44 0.5:1   99.50 99.3999.33 98.76 96.77 90.07 78.03 65.63 54.07 0.2:1   99.46 99.37 99.3398.24 96.50 85.72 69.89 55.13 41.97 0:1 99.44 99.39 99.12 97.28 93.3159.45 37.09 23.57 15.48

TABLE 11 MgCl₂:Tygacil ® Molar ratio Day 0 Day 1 Day 2 Day 5 Day 7 Day14 Day 21 Day 28 Day 36 10:1  99.44 99.53 99.34 99.25 99.07 97.30 95.3792.20 86.32 5:1 99.44 99.61 99.60 99.45 99.32 97.66 95.34 90.98 83.582:1 99.48 99.63 99.56 99.43 99.19 96.67 91.94 81.95 66.57 1:1 99.5599.62 99.61 99.09 99.11 96.50 89.71 74.36 55.95 0.5:1   99.49 99.6499.60 99.33 98.70 95.10 84.39 64.70 45.04 0.2:1   99.49 99.63 99.5799.28 98.89 94.03 79.53 57.09 37.94 0:1 99.44 99.57 99.57 99.25 98.7889.19 65.09 42.56 28.38

TABLE 12 CaCl₂:Tygacil ® Molar ratio Day 0 Day 1 Day 2 Day 5 Day 7 Day14 Day 21 Day 28 Day 36 10:1  99.32 99.51 99.45 99.50 99.26 97.41 95.0892.06 87.88 5:1 99.35 99.51 — 99.33 99.02 97.36 93.42 88.57 82.75 2:199.40 99.67 99.46 99.25 98.97 95.76 90.00 81.77 72.75 1:1 99.49 99.6099.54 99.39 99.02 95.44 88.25 77.42 65.65 0.5:1   99.48 99.60 99.4999.30 98.55 94.80 85.57 71.96 58.07 0.2:1   99.44 99.57 99.53 99.2798.89 92.70 80.03 62.28 47.05 0:1 99.45 99.60 99.55 99.18 98.70 88.0263.58 40.77 28.00

TABLE 13 ZnCl₂:Tygacil ® Molar ratio Day 0 Day 1 Day 2 Day 5 Day 7 Day14 Day 21 Day 28 Day 36 10:1  98.91 99.49 99.43 99.46 99.47 98.98 98.6898.17 98.11 5:1 99.15 99.54 99.51 99.45 99.35 98.88 98.26 97.39 96.152:1 99.29 99.57 99.55 99.35 99.37 98.60 97.42 95.30 92.37 1:1 99.4499.62 99.55 99.61 99.33 97.97 96.29 92.70 87.08 0.5:1   99.47 99.6299.59 99.48 99.25 97.60 94.10 86.46 76.49 0.2:1   99.45 99.62 99.6199.47 99.19 96.09 89.52 77.46 63.06 0:1 99.42 99.54 99.52 99.14 98.7188.25 64.08 41.19 28.09

While Tigecycline decomposed in all tubes over 36 days, the rate ofdecomposition was significantly lower in solutions containing highermolar ratios of metal cation. The rates of Tigecycline decomposition inthe presence of calcium or magnesium cations were similar; however, therate of Tigecycline decomposition in the presence of zinc wassignificantly lower. The presence of lactose in the Tygacil® formulationfurther decreased the rate of decomposition.

Example 3 Stability at 37° C. for Tygacil Solutions Containing HighConcentrations of Metal Cations

A 10 mg/mL aqueous solution of Tygacil® (Lot D 90293, 53 mg) wasprepared, and 300 μL aliquots were dispensed into polypropylene tubes.The volume of each tube was adjusted to 1 ml with various dilutions of 1M MgCl₂, 1 M CaCl₂ or 1 M ZnCl₂ to achieve the desired molar ratio ofTigecycline:metal cation. The tubes were incubated in the dark at 37° C.Samples of each solution were taken at various time points and analyzedby HPLC. The fraction of remaining Tigecycline in each sample wasdetermined.

The percentages of Tigecycline remaining at Day 0, 1, 2, 5, 7, 14, and21 for solutions of Tygacil® at various ratios with MgCl₂, CaCl₂, orZnCl₂ are shown in TABLE 14, TABLE 15, and TABLE 16, respectively.

TABLE 14 MgCl₂:Tygacil ® Day Day Day Day Day Molar ratio Day 0 Day 1 2 57 14 21 30:1 99.64 99.59 99.49 98.54 97.11 89.62 77.13 20:1 99.61 99.5699.23 97.99 95.94 85.04 63.47 12:1 99.58 99.53 99.14 96.74 94.45 77.7146.81  5:1 99.68 99.56 99.6 96.06 91.18 59.13 25.95  0:1 99.65 99.2398.26 75.05 46.66 6.37 1.30

TABLE 15 CaCl₂:Tygacil ® Day Day Day Day Day Molar ratio Day 0 Day 1 2 57 14 21 30:1 99.58 99.55 99.29 97.79 95.9 86.94 69.71 20:1 99.62 99.5199.18 97.00 93.81 80.6 55.28 12:1 99.60 99.41 98.94 94.94 91.13 69.340.59  5:1 99.65 99.42 98.66 92.83 85.72 53.1 24.74  0:1 99.60 99.3498.25 74.61 45.63 6.26 1.53

TABLE 16 ZnCl₂:Tygacil ® Molar Day Day Day Day Day ratio Day 0 Day 1 2 57 14 21 12:1  99.44 99.27 99.49 98.60 97.66 92.50 83.58 5:1 99.48 —99.22 97.42 96.21 87.22 71.55 0:1 99.62 — 98.22 73.43 43.3 6.37 1.57

While Tigecycline decomposed in all tubes over 21 days, the rate ofdecomposition was significantly lower in solutions containing highermolar ratios of metal cation. The rates of Tigecycline decomposition inthe presence of calcium or magnesium cations were similar, however, therate of Tigecycline decomposition in the presence of zinc wassignificantly lower.

Example 4 Effect of pH on the Stability of Tygacil® Solutions ContainingMetal Cations at 37° C.

A 10 mg/mL aqueous solution of Tygacil® (Lot D 90293, 53 mg) wasprepared, and 1650 μL aliquots were dispensed into four 15 mLpolypropylene tubes. The volume of each tube was adjusted to 5500 μLwith various dilutions of 0.1 M MgCl₂, 0.1 M CaCl₂, or 0.1 M ZnCl₂, orwater (control), to achieve the desired molar ratio of a 1:1 ratio ofTigecycline: metal cation. Sample solutions from each 15 ml tube weretaken and adjusted to pH 4, 5, or 6 with 0.1 N or 1 N solutions of NaOHor HCl, taking care to minimize volume changes. Samples solutions wereincubated in the dark at 37° C. Samples were taken at various timepoints and analyzed by HPLC. The fraction of remaining Tigecycline(expressed as a percentage of the starting concentration) in each samplewas determined.

The percentages of Tigecycline remaining at Day 0, 1, 2, 5, 7, and 14for solutions of Tygacil® at 1:1 ratios with MgCl₂, CaCl₂, or ZnCl₂ atvarious pHs are shown in TABLE 17, TABLE 18, and TABLE 19, respectively.TABLE 20 shows percentages of Tigecycline remaining at Day 0, 1, 2, 5,7, and 14 for solutions of Tygacil® only at various pHs

TABLE 17 pH for 1:1 Day MgCl₂:Tygacil ® Day 0 Day 1 Day 2 Day 5 Day 7 14pH 4 99.51 98.89 98.89 95.72 90.92 54.54 pH 5 99.55 99.09 98.00 84.7763.60 15.89 pH 6 99.53 98.36 95.79 44.81 23.71 5.19

TABLE 18 pH for 1:1 Day CaCl₂:Tygacil ® Day 0 Day 1 Day 2 Day 5 Day 7 14pH 4 99.49 98.88 98.84 94.43 90.06 55.91 pH 5 99.66 99.02 97.8 81.9669.23 28.89 pH 6 99.62 98.70 97.87 92.45 87.40 56.79

TABLE 19 pH for 1:1 ZnCl₂: Day Day Day Day Day Day Tygacil ® 0 1 2 5 714 pH 4 99.47 98.62 99.03 96.14 93.15 73.25 pH 5 99.6 99.21 98.96 93.0283.48 39.93 pH 6 99.54 99.3 99.16 94.58 86.35 49.21

TABLE 20 pH for Day Day Day Day Day Day Tygacil ® 0 1 2 5 7 14 pH 499.48 99.07 98.93 94.28 87.05 44.75 pH 5 99.6 98.94 96.89 49.21 27.492.16 pH 6 99.47 95.27 59.56 10.74 2.4 5.22

While Tigecycline decomposed in all tubes over 14 days, the rate ofdecomposition was significantly lower in solutions with a pH lower thanpH 6. The rates of Tigecycline decomposition in the presence of calciumor magnesium cations were similar at pH 4 and 5; however, the rate ofTigecycline decomposition in the presence of magnesium at pH 6 wassignificantly greater. The rate of Tigecycline decomposition at pH 4 and5 in solutions containing zinc was lower than solutions containingmagnesium or calcium. The rates of Tigecycline decomposition at pH 6, insolutions containing zinc or calcium were similar. The rate ofTigecycline decomposition at all pHs was much lower in the presence ofmetal cations, especially at higher pH.

Example 5 Effect of pH on the Stability of Tygacil® Solutions ContainingHigh Concentrations of Metal Cations at 37° C.

A 10 mg/mL aqueous solution of Tygacil® (Lot D 90293, 53 mg) wasprepared, and 1650 μL aliquots were dispensed into four 15 mLpolypropylene tubes. The volume of each tube was adjusted to 5500 μLwith various dilutions of 1 M MgCl₂, 1 M CaCl₂, or 1 M ZnCl₂, or water(control), to achieve the desired molar ratio of a 1:12 ratio ofTigecycline:metal cation. Sample solutions from each 15 ml tube weretaken and adjusted to pH 4, 5, or 6 with 0.1 N or 1 N solutions of NaOHor HCl, taking care to minimize volume changes. Samples solutions wereincubated in the dark at 37° C. Samples were taken at various timepoints and analyzed by HPLC. The fraction of remaining Tigecycline ineach sample was determined.

The percentages of Tigecycline remaining at Day 0, 1, 2, 5, 7, and 14for solutions of Tygacil® at 1:12 ratios with MgCl₂, CaCl₂, or ZnCl₂ atvarious pHs are shown in TABLE 21, TABLE 22, and TABLE 23, respectively.

TABLE 21 pH for 12:1 MgCl₂: Day Day Day Day Day Day Tygacil ® 0 1 2 5 714 pH 4 99.47 98.62 99.18 97.49 95.72 83.14 pH 5 99.61 98.87 99.12 96.5393.72 69.08 pH 6 99.58 99.26 99.21 95.6 96.96 85.86

TABLE 22 pH for 12:1 CaCl₂: Day Day Day Day Day Day Tygacil ® 0 1 2 5 714 pH 4 99.48 97.24 98.89 96.01 92.85 73.05 pH 5 99.74 99.36 99.41 97.6495.94 89 pH 6 99.61 99.44 99.48 98 97.09 92.18

TABLE 23 pH for 12:1 ZnCl₂: Day Day Day Day Day Day Tygacil ® 0 1 2 5 714 pH 4 99.49 99.29 99.36 98.73 98.35 95.19 pH 5 99.56 99.47 99.47 98.3898.04 93.38 pH 6 99.65 99.38 99.49 98.78 98.79 97.67

While tigecycline decomposed in all tubes over 14 days, the rate ofdecomposition was slower in solutions at pH 6. The rates of Tigecyclinedecomposition in the presence of calcium were slower in solutions atgreater pH. When formulated as Tygacil, the rates of tigecyclinedecomposition in the presence of zinc or magnesium were faster at pH 5.

Example 6 Effect of pH on the Stability of Minocycline SolutionsContaining High Concentrations of MgCl₂ at 37° C.

A 10 mg/mL Minocycline hydrochloride aqueous solution was prepared, and2500 μL aliquots were dispensed into two 15 mL polypropylene tubes. Thevolume of each tube was adjusted to 5500 μL with either a dilution of 1M MgCl₂ to achieve a molar ratio of a 1:10 ratio of Minocycline:metalcation, or water. Sample solutions from each 15 ml tube were taken andadjusted to pH 4, 5, or 6 with 0.1 N or 1 N solutions of NaOH or HCl,taking care to minimize volume changes. Sample solutions were incubatedin the dark at 37° C. Samples were taken at various time points andanalyzed by HPLC. The fraction of minocycline remaining in each samplewas determined.

The percentages of Minocycline remaining at Day 0, 1, 2, 5, 7, and 14for solutions at various pHs of Minocycline at 1:10 ratio with MgCl₂, orMinocycline solutions alone are shown in TABLE 24, and TABLE 25,respectively.

TABLE 24 pH for 10:1 MgCl₂: Day Day Day Day Day Day Minocycline 0 1 2 57 14 pH 4 98.63 96.97 96.46 94.76 93.43 84.32 pH 5 98.69 97.05 96.1993.01 89.31 75.42 pH 6 99.03 97.1 96.04 88.45 83.88 76.25

TABLE 25 pH for Minocycline Day Day Day Day Day Day alone 0 1 2 5 7 14pH 4 98.75 96.37 96.21 94.99 92.78 81.82 pH 5 98.41 96.72 95.29 85.0175.14 35.43 pH 6 98.19 95.47 87.55 39.17 14.56 2.2

While Minocycline decomposed in all tubes over 14 days, the rate ofdecomposition was significantly lower in solutions containing magnesium,especially at higher pH.

Example 7 Stability of Tigecycline Solutions Containing Mixtures ofCaCl₂ and MgCl₂ at pH 6 and 37° C.

A 10 mg/mL aqueous solution of Tigecycline was prepared, and 450 ptaliquots were dispensed into 15 mL polypropylene tubes. The volume ofeach tube was adjusted to 1500 μL with various dilutions of 1 M MgCl₂and 1 M CaCl₂, or water (control), to achieve the desired molar ratiosof Tigecycline:metal cation. Sample solutions from each 15 ml tube weretaken and adjusted to pH 6 with 0.1 N or 1 N solutions of NaOH or HCl,taking care to minimize volume changes. Samples solutions were incubatedin the dark at 37° C. Samples were taken at various time points andanalyzed by HPLC. The fraction of Tigecycline remaining in each samplewas determined.

The percentages of Tigecycline remaining at Day 0, 1, 2, 5, 7, 14, and21 for solutions of at various ratios of Tigecycline: MgCl₂: CaCl₂ at pH6 are shown in TABLE 26.

TABLE 26 MgCl₂:CaCl₂: tigecycline Day Day Day Day Day Day Day Molarratio 0 1 2 5 7 14 21 5:5:1 98.25 98.77 98.23 96.91 92.13 83.64 65.215:10:1 98.37 98.23 98.59 97.76 96.10 89.74 79.83 10:5:1 98.17 98.2198.46 96.59 93.90 80.00 59.39 10:10:1 98.32 98.24 98.50 97.38 95.6287.14 72.88 5:0:1 98.18 97.93 97.53 90.58 76.71 40.42 12.54 10:0:1 98.1698.00 98.23 94.91 89.12 62.54 35.75 15:0:1 98.25 98.13 98.21 96.23 92.3272.15 48.75 20:0:1 98.2 98.08 98.28 96.46 93.72 78.66 57.66 0:5:1 98.1198.15 98.28 97.19 95.68 89.2 77.2 0:10:1 98.12 98.2 98.55 97.1 96.5391.74 84.69 0:15:1 98.15 98.21 98.59 97.5 96.93 92.71 86.37 0:20:1 98.2898.63 98.57 97.4 97.35 93.09 87.45 0:0:1 97.91 88.97 60.59 16.36 7.334.14 0

While Tigecycline decomposed in all tubes over 21 days, the rate ofdecomposition was significantly lower in solutions containing greaterrelative amounts of calcium cations.

Example 8 Effects of MgCl₂ on Minocyline-Induced Hemolysis in an InVitro Model of Venous Phlebitis

In vitro hemolysis of rabbit red blood cells (RBCs) after exposure tominocycline formulated in MgCl₂ or CaCl₂ was compared to in vitrohemolysis of RBCs after exposure to minocycline in saline, or exposureto amphotericin B. Minocycline HCl (LKT laboratories) stock solutionswere prepared with MgCl₂ in saline, saline, or lactated ringer, and thepH was adjusted with NaOH. Rabbit and sheep red blood cells (RBCs) wereobtained from Innovative Research laboratory (Michigan, USA).Immediately before use, RBCs were washed three times in 0.9% saline andadjusted to a density of 5% in saline. 200 μl RBCs was added to 800 μlminocycline solution, and mixed by gentle inversion for 2-5 seconds.Samples were incubated at 37° C. or 30 minutes or at 25° C. for 2-5minutes. Incubated samples were centrifuged at 12000×g for 4 minutes andthe supernatants were removed and the hemoglobin absorbance was read at540 nm. Samples were tested in triplicate. Amphotericin B (MPBiomedicals) and distilled H₂O, or Triton-x and distilled H₂O were usedas positive controls; saline was used as a negative control. Percenthemolysis was calculated according to the following formula:

${{Percent}\mspace{14mu}{hemolysis}} = {\frac{( {{absorbance}\mspace{14mu}{of}\mspace{14mu}{sample}} ) - ( {{absorbance}\mspace{14mu}{of}\mspace{14mu}{blank}} )}{{Absorbance}\mspace{14mu}{of}\mspace{14mu}{Distilled}\mspace{14mu} H_{2}O} \times 100}$

In a set of experiments, the pH of minocycline solutions formulated withdivalent cations was adjusted to pH 5.85. For RBCs incubated in aminocycline saline solution, hemolysis was in the range of 44%-84% (FIG.1). For RBCs incubated in a minocycline with Mg²⁺ or Ca²⁺, hemolysis wasapproximately 2%. Results summarizing the percent in vitro hemolysis ofrabbit RBCs incubated with different formulations of minocycline oramphoterin B at 25° C. are summarized in Table 27.

TABLE 27 Hemolysis of RBCs in solution relative Solution to water (%) 5mg/ml minocycline, 10 equiv Mg, pH 5.85 2.8 2.5 mg/ml minocycline, 10equiv Mg, pH 3.2 5.85 0.5 mg/ml minocycline, 10 equiv Mg, pH 2.3 5.85 5mg/ml minocycline, 5 equiv Ca, pH 5.85 2.2 2.5 mg/ml minocycline, 5equiv Ca, pH 5.85 2.94 0.5 mg/ml minocycline, 5 equiv Ca, pH 5.85 2.20 5mg/ml minocycline, saline, pH 4.17 81.64 2.5 mg/ml minocycline, saline,pH 4.17 84.37 0.5 mg/ml minocycline, saline, pH 4.17 43.82 Amphoterin B101.31

In another set of experiments, the pH of a minocycline solutionformulated with divalent cations was not adjusted and was allowed tofall below the pH of minocycline in saline. For RBCs incubated in aminocycline saline solution, hemolysis was in the range of 44%-84% (FIG.2). For RBCs incubated in a minocycline with Mg²⁺ or Ca²⁺, hemolysis wasin the range of 0%-5%. Results summarizing the percent in vitrohemolysis of rabbit RBCs incubated with different formulations ofminocycline at low pH, or amphoterin B at 25° C. are summarized in Table28.

TABLE 28 Hemolysis of RBCs in solution relative Solution to water (%) 5mg/ml minocycline, 10 equiv Mg, pH 3.5 0.88 2.5 mg/ml minocycline, 10equiv Mg, pH 1.12 3.5 0.5 mg/ml minocycline, 10 equiv Mg, pH 2.20 3.5 5mg/ml minocycline, 5 equiv Ca, pH 3.64 — 2.5 mg/ml minocycline, 5 equivCa, pH 0.86 3.64 0.5 mg/ml minocycline, 5 equiv Ca, pH 4.92 3.64 5 mg/mlminocycline, saline, pH 4.17 81.64 2.5 mg/ml minocycline, saline, pH4.17 84.37 0.5 mg/ml minocycline, saline, pH 4.17 43.82 Amphoterin B101.31

Hemolysis of RBCs was reduced in an in vitro model of venous phlebitiswith minocycline solutions formulated with divalent cations compared tominocycline solutions formulated without divalent cations.

In another set of experiments, hemolysis of rabbit RBCs was measuredafter exposure to 2.5 mg/ml minocycline formulated with differentrations of divalent cations (MgCl₂, Mg SO₄, or CaCl₂). Hemolysis wascompared to Minocycline HCl; Triton-x and H₂O were used as positivecontrols. Results are summarized in Table 29 and shown in FIGS. 4-6.

TABLE 29 2.5 mg/ml minocycline solution Hemolysis of RBCs Molar ratio insolution relative Cation cation:minocycline to water (%) MgSO₄ 1:2 22.521:1 24.59 2:1 40.87 3:1 25.67 5:1 2.86 7:1 1.96 10:1  0.19 MgCl₂ 1:246.91 1:1 63.77 2:1 74.87 3:1 64.62 5:1 9.43 7:1 1.57 10:1  0.35 CaCl₂1:2 75.22 1:1 83.89 2:1 50.84 3:1 26.58 5:1 1.16 7:1 0.75 10:1  0.40Minocycline only 37.44 Triton-x 97.82

FIG. 3 and FIG. 4 show the degree of rabbit RBC hemolysis produced byminocycline formulated in different ratios of MgSO₄ or MgCl₂,respectively, compared to Minocycline only. The data indicates that a5:1 molar ratio of magnesium to minocycline or greater inhibits the RBChemolysis observed with minocycline alone. Minocycline (minocin)produced a relative RBC hemolysis of 37%. FIG. 5 shows the degree ofrabbit RBC hemolysis produced by minocycline formulated in differentratios of CaCl₂. This data shows that a 5:1 molar ratio of calcium tominocycline inhibits the RBC hemolysis observed with minocycline HClalone.

Overall, these data all suggest that high molar ratios (e.g., a 5:1molar ratio or greater) of divalent cation (Mg⁺² or Ca⁺²) to minocyclineresults in significant inhibition of rabbit RBC hemolysis observed withminocycline HCl.

Example 9 Solubility of Minocycline with Divalent Cations

Mixtures were prepared containing minocycline and divalent cations (Mg²⁺or Ca²⁺) at varying stoichiometry and pH. The solubility of minocyclinewas assessed according to the turbidity of the mixture at 0 hr, 24 hr,48 hr, 72 hr, 96 hr, 120 hr, 144 hr, and 168 hr. A clear solutiondenoted complete solubility. Table 30 summarizes data for minocyclinewith Mg²⁺ at 0 hr and 24 hr. Table 31 summarizes data for minocyclinewith Ca²⁺ at 0 hr and 24 hr.

TABLE 30 Molar ratio cation (Mg²⁺):Minocycline 0 1:2 1:1 2:1 3:1 5:1 7:110:1 Time (hr) 0 24 0 24 0 24 0 24 0 24 0 24 0 24 0 24 1 mg/ml pH 4 ∘ ∘∘ ∘ ∘ ∘ ∘ ∘ minocycline pH 5 ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ pH 6 ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ pH 7 ∘∘ ∘ ∘ ∘ ∘ ∘ ∘ 5 mg/ml pH 4 ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ minocyclinepH 5 ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ pH 6 ∘ ∘ • • • • • • • • ∘ • ∘ • ∘∘ 10 mg/ml pH 4 ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ minocycline pH 5 ∘ ∘ ∘ ∘∘ • ∘ • ∘ • ∘ ∘ ∘ ∘ ∘ ∘ pH 6 ∘ ∘ • • • • • • • • • • • • • • 20 mg/ml pH4 ∘ • ∘ ∘ ∘ ∘ ∘ ∘ minocycline pH 5 ∘ ∘ ∘ • ∘ • ∘ • 30 mg/ml pH 4 ∘ • ∘ ∘∘ ∘ ∘ ∘ minocycline pH 5 ∘ • ∘ • ∘ • ∘ • •: insoluble; ∘: soluble

TABLE 31 Molar ratio cation (Ca²⁺):minocycline 0 1:2 1:1 2:1 3:1 5:1 7:110:1 Time (hr) 0 24 0 24 0 24 0 24 0 24 0 24 0 24 0 24 1 mg/ml pH 4 ∘ ∘∘ ∘ ∘ ∘ ∘ ∘ minocycline pH 5 ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ pH 6 ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ pH 7 ∘∘ ∘ • ∘ • ∘ • 5 mg/ml pH 4 ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ minocyclinepH 5 ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ pH 6 ∘ ∘ • ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘∘ 10 mg/ml pH 4 ∘ ∘ ∘ ∘ ∘ ∘ minocycline pH 5 ∘ ∘ ∘ ∘ ∘ ∘ pH 6 ∘ ∘ ∘ ∘ ∘∘ 20 mg/ml pH 4 ∘ • ∘ ∘ ∘ ∘ ∘ ∘ minocycline pH 5 ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ 30mg/ml pH 4 ∘ • ∘ • ∘ • ∘ • minocycline pH 5 ∘ • ∘ ∘ ∘ ∘ ∘ ∘ •:insoluble; ∘: soluble

The data demonstrates that minocycline stays in solution uponintroduction of a cation at concentrations of 10 mg/ml and less if thepH is less than 5. At higher pH, introduction of a cation initiallyreduces solubility. For example, a 5 mg/ml minocycline solution at pH 6becomes insoluble on addition of Mg²⁺. Surprisingly, at a molar ratio ofcation:minocycline of 5:1 or more, the minocycline of such solutionsbecomes soluble, suggesting that high ratios of cation increases thesolubility of minocycline.

Table 32 summarizes data for minocycline with Mg²⁺ at 48 hr and 72 hr.

TABLE 32 Molar ratio cation (Mg²⁺):minocycline 0 1:2 1:1 2:1 3:1 5:1 7:110:1 Time (hr) 48 72 48 72 48 72 48 72 48 72 48 72 48 72 48 72 1 mg/mlpH 4 ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ minocycline pH 5 ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ pH 6 ∘ ∘ ∘ ∘ ∘ ∘ ∘∘ pH 7 ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ 5 mg/ml pH 4 ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘minocycline pH 5 ∘ ∘ ∘ • ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ pH 6 ∘ ∘ • • • • • • •• • • • • • • 10 mg/ml pH 4 • ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ minocyclinepH 5 • • ∘ • • • • • • • ∘ • ∘ • ∘ ∘ pHm 6 • • • • • • • • • • • • • • •• 20 mg/ml pH 4 • • ∘ ∘ ∘ ∘ ∘ ∘ minocycline pH 5 • • • • • • • • 30mg/ml pH 4 • • ∘ ∘ ∘ ∘ ∘ ∘ minocycline pH 5 • • • • • • • • •:insoluble; ∘: soluble

Example 10 Long-Term Stability of Tigecycline at Various Temperatures

Table 33, Table 34, and Table 35 show percentage remaining tigecyclinefor different formulations of tigecycline at pH 6, stored at 37° C.,room temperature, and 4° C., respectively. Formulations of tigecyclinecomprising increasing concentrations of tigecycline and increasingconcentrations of CaCl₂ showed increased stability.

TABLE 33 Formu- lation Stability of tigecycline (%) stored 0 1 2 5 7 14Salt at 37° C. day day days days days days MgCl₂ 12 eq 20 97.97 97.4396.37 92.63 88.41 mg/mL 5 eq 20 98.09 97.38 96.42 88.64 81.62 mg/mL 2 eq20 97.95 97.28 94.1 80.59 69.88 mg/mL 12 eq 3 98.17 98.05 97.08 93.7888.16 mg/mL 5 eq 3 98.3 97.72 96.77 86.97 73.76 mg/mL 2 eq 3 98.21 97.2293.75 62.21 45.31 mg/mL CaCl₂ 12 eq 20 98.3 98 97.63 96.1 95.24 91.44mg/mL 5 eq 20 98.16 97.75 97.4 95.82 94.81 89.26 mg/mL 2 eq 20 98.2597.85 97.22 95.28 93.64 88.61 mg/mL 12 eq 3 98.29 98.03 97.74 96.7995.92 91.07 mg/mL 5 eq 3 98.21 97.96 97.32 95.37 94.42 86.36 mg/mL 2 eq3 98.17 97.74 96.57 92.99 90.22 mg/mL ZnCl₂ 1 eq 20 98.26 97.19 93.8681.02 72.41 mg/mL 1 eq 3 98.29 97.88 96.73 86.5 74.32 mg/mL

TABLE 34 Formu- lation stored at room Stability of tigecycline (%)temper- 0 7 14 28 42 58 Salt ature day days days days days days MgCl₂ 12eq 20 97.97 96.56 93.4 79.44 mg/mL 5 eq 20 98.09 94.2 82.17 mg/mL 2 eq20 97.95 87.57 67.91 mg/mL 12 eq 3 98.17 97.22 94.91 80.14 mg/mL 5 eq 398.3 96.45 89.91 mg/mL 2 eq 3 98.21 92.66 66.91 mg/mL CaCl₂ 12 eq 2098.3 97.91 97.36 95.69 95.32 93.02 mg/mL 5 eq 20 98.16 97.88 97.23 95.2494.08 90.78 mg/mL 2 eq 20 98.25 97.97 97.08 94.42 93.08 87.95 mg/mL 12eq 3 98.29 98.01 97.7 96.37 95.78 93.67 mg/mL 5 eq 3 98.21 97.84 97.2995.39 94.22 90.37 mg/mL 2 eq 3 98.17 97.53 96.47 92.85 90.07 79.52 mg/mLZnCl₂ 1 eq 20 98.26 82.44 65.73 mg/mL 1 eq 3 98.29 97.11 93.1 mg/mL

TABLE 35 Formu- lation Stability of tigecycline (%) stored 0 14 28 35 58162 Salt at 4° C. day day days days days days MgCl₂ 12 eq 97.97 97.6896.16 95.36 89.95 20 mg/mL 5 eq 20 98.09 96.22 78.05 69.76 mg/mL 2 eq 2097.95 91.23 54.38 43.33 mg/mL 12 eq 3 98.17 97.76 95.76 94.19 80.31mg/mL 5 eq 3 98.3 97.48 91.75 86.21 mg/mL 2 eq 3 98.21 96.23 84.6 76.81mg/mL CaCl₂ 12 eq 98.3 98.28 97.78 97.61 97.87 96.4 20 mg/mL 5 eq 2098.16 97.97 97.65 97.79 97.78 95.22 mg/mL 2 eq 20 98.25 98.08 97.69 97.897.75 94.9 mg/mL 12 eq 3 98.29 98.37 98.16 97.79 98.15 97.27 mg/mL 5 eq3 98.21 98.17 97.97 97.76 97.99 96.75 mg/mL 2 eq 3 98.17 98.14 97.4597.53 97.56 93.35 mg/mL ZnCl₂ 1 eq 20 98.26 77.12 53.06 45.63 mg/mL 1 eq3 98.29 97.73 96.38 95.02 88.53 mg/mL

Example 11 Solubility of Tetracycline Formulations

The solubility of four non-dimethlyamino tetracyclines, with and withoutMg²⁺, was examined. The results are summarized in Table 36.

TABLE 36 Molar ratio cation (Mg²⁺):antibiotic 0 0.5:1 1:1 2:1 3:1 5:17:1 10:1 10 mg/ml pH 4 • • • • • • • • tetracycline pH 5 • • • • • • • •pH 6 • • • • • • • • 10 mg/ml pH 4 • ∘ ∘ ∘ chlortetracycline pH 5 • • •• pH 6 • • • • 10 mg/ml pH 4 ∘ • • • doxycycline pH 5 ∘ • • • pH 6 ∘^(#)∘^(#) ∘^(#) 10 mg/ml pH 4 • • • • oxytetracycline pH 5 • • ∘ ∘ pH 5 • •∘ ∘ •: insoluble; ∘: soluble; ^(#)fell out of solution after 24 hrs atroom temperature

A comparison with the results for minocycline described in Example 9indicates that non-dimethylamino-tetracylines, such as tetracycline,chlortetracycline, doxycycline, and oxytetracycline have solubilitycharacteristics that differ from dimethylamino-tetracylines. Forexample, as summarized in Table 36, tetracycline remains insoluble atvarious pH and amounts of a divalent cation such as Mg²⁺.Chlortetracycline becomes soluble with increasing concentrations of adivalent cation, but remains insoluble in the absence of any divalentcation, such as Mg²⁺. Doxycycline is soluble in the absence of divalentcations, such as Mg²⁺, but is insoluble in the presence of divalentcations at low pH. Similarly, oxytetracycline remains insoluble in thepresence of divalent cations, such as Mg²⁺, at low pH.

Example 12 Study of the Effect of Mg²⁺ on the Uptake of Minocycline inHuman Umbilical Vein Endothelial Cells (HUVEC)

Cells and reagents: Human umbilical vein endothelial cells (HUVEC) werepurchased from Lonza and maintained according to manufacturer'srecommendations in EGM-2 media. A 10 mg/mL solution of minocycline wasprepared in 13.6 mg/mL Na-acetate without addition of Mg. This stocksolution was further diluted in saline to 1 mg/mL with addition of Mg inthe form of 1 M MgSO₄ to generate the following molar ratios of Mg tominocycline: 0, 1, 2.5, 5, 10, 25.

Uptake experimental conditions: HUVECs were seeded at 4.5×10⁵ cells/welldensity in 6-well plates in EGM-2 media. Two days after seeding, cellswere washed once with 2 mL of saline, and then 2 mL of 1 mg/mL drugsolution in saline prepared as described above was placed in each wellin triplicate. Plates were incubated in a CO₂ incubator at 37° C. for 30min. Drug solutions were aspirated and cells were washed once with 2 mLof saline. 0.5 mL of saline was placed in each well and the cellmonolayer was scraped using a plastic cell scraper. Cell suspensionswere transferred to 1.5 mL plastic tubes and sonicated for 30 sec atmaximal power. Cell lysates were spun down for 10 min on a table topmicrocentrifuge at maximum speed and supernatants were collected.Several wells of HUVEC cells were treated with saline only and processedthe same way as drug-treated cells to generate mock cell lysate whichwas used below for calibration curve preparation.

Sample preparation for LCMS analysis: To prepare a calibration curve, 1mg/mL minocycline solution in water was diluted in mock cell lysate toproduce 100 μl of standards with the following concentrations: 10, 5, 2,1, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01 μg/ml.

50 μl of supernatants from drug-treated samples or standards were mixedwith 200 μ1 of 1% trifluoroacetic acid in acetonitrile containing 1μg/mL of gatifloxacin, vortexed and centrifuged at 3000 g for 30 min atRT. 150 μl of supernatants was removed and mixed with 450 μl of water.After vortexing, the mixture was centrifuged at 3000 g for 5 min at RT.Supernatants were collected and subjected to LCMS analysis to determineminocycline concentration.

Data processing: Uptake data were presented as percentage relative tothe sample with no Mg present, which was considered as 100%.

Uptake of minocycline at 1 mg/mL in saline with various Mg/minocyclineratios was tested in HUVEC with an incubation time was 30 min. Theresults are summarized in FIG. 6 and FIG. 7. FIGS. 6 and 7 demonstratethat a decrease in intracellular uptake of minocycline is observed asthe concentration of a divalent cation, such as Mg²⁺ increases. Whilenot being bound by any particular theory, this result suggests that themechanism for the reduction in hemolysis observed in theminocycline/cation formulations described herein may be attributed toreduced RBC updake.

Example 13 Preparing Certain Formulations of Dimethylamino-Tetracylines

Formulation 1

A formulation comprising minocycline with MgCl₂ and NaOH suitable forintravenous administration is prepared. 100 mg minocycline is added to a10 ml aqueous solution of MgCl₂.6H₂O to provide a cation to minocyclinemolar ratio of 5:1 and a 10 mg/ml minocycline solution. The pH of themixture is adjusted by adding NaOH to a pH in the range of pH 4.5-pH5.5. A single attempt of lyophilization resulted in a non-flocculentsolid.

Formulation 2

A formulation comprising minocycline with MgSO₄ and sodium acetatesuitable for intravenous administration is prepared. 100 mg minocyclineis added to an aqueous solution of MgSO₄.7H₂O to provide a cation tominocycline molar ratio of 5:1 and a 10 mg/ml minocycline solution. ThepH of the solution is adjusted by adding sodium acetate to a pH in therange of pH 4.5-pH 5.5. The solution is then lyophilized to dryness.Reconstitution of the lyophile in 10 ml water results in a solutionhaving a pH in the range of pH 4.5-pH 5.5 and an osmolality in the rangeof 275 mOsm/kg-375 mOsm/kg.

Formulation 3

A formulation comprising minocycline with Mg(C₂H₃O₂)₂ suitable forintravenous administration is prepared. 100 mg minocycline is added toan aqueous solution of Mg(C₂H₃O₂)₂.3H₂O to provide a cation tominocycline molar ratio of 5:1 and a 10 mg/ml minocycline solution. Thesolution is then lyophilized to dryness.

Formulation 4

A formulation comprising minocycline with MgSO₄ and NaOH suitable forintravenous administration is prepared. 100 mg minocycline is added toan aqueous solution of MgSO₄.7H₂O to provide a cation to minocyclinemolar ratio of 5:1 and a 10 mg/ml minocycline solution. The pH of thesolution is adjusted by adding NaOH to a pH in the range of pH 4.5-pH5.5. The solution is lyophilized to dryness. Reconstitution of thelyophile in 10 ml water results in a solution having a pH in the rangeof pH 4.5-pH 5.5 and an osmolality in the range of 150 mOsm/kg-250mOsm/kg.

Formulation 5

A formulation comprising tigecycline with MgSO₄ and NaOH suitable forintravenous administration is prepared. 50 mg tigecycline is added to 10ml aqueous solution of MgSO₄.7H₂O to provide a cation to tigecyclinemolar ratio of 5:1. The pH of the solution is adjusted by adding NaOH toa pH in the range of pH 5.5-pH 6.5. The solution is then lyophilized todryness. Reconstitution of the lyophile in 10 ml water results in asolution having a pH in the range of pH 5.5-pH 6.5.

Formulation 6

A formulation comprising tigecycline with MgSO₄ and NaOH suitable forintravenous administration is prepared. 50 mg tigecycline is added to 10ml aqueous solution of MgSO₄.7H₂O to provide a cation to tigecyclinemolar ratio of 12:1. The pH of the solution is adjusted by adding NaOHto a pH in the range of pH 5.5-pH 6.5. The solution is then lyophilizedto dryness. Reconstitution of the lyophile in 10 ml water results in asolution having a pH in the range of pH 5.5-pH 6.5.

Formulation 7

A formulation comprising tigecycline with MgCl₂ and NaOH suitable forintravenous administration is prepared. 50 mg tigecycline is added to 10ml aqueous solution of MgCl₂.6H₂O to provide a cation to tigecyclinemolar ratio of 5:1. The pH of the solution is adjusted by adding NaOH toa pH in the range of pH 5.5-pH 6.5. The solution is then lyophilized todryness. Reconstitution of the lyophile in 10 ml water results in asolution having a pH in the range of pH 5.5-pH 6.5.

Formulation 8

A formulation comprising tigecycline with MgCl₂ and NaOH suitable forintravenous administration is prepared. 50 mg tigecycline is added to 10ml aqueous solution of MgCl₂.6H₂O to provide a cation to tigecyclinemolar ratio of 12:1. The pH of the solution is adjusted by adding NaOHto a pH in the range of pH 5.5-pH 6.5. The solution is then lyophilizedto dryness. Reconstitution of the lyophile in 10 ml water results in asolution having a pH in the range of pH 5.5-pH 6.5.

Formulation 9

A formulation comprising tigecycline with MgSO₄ and NaOH suitable fortopical administration is prepared. 50 mg tigecycline is added to 10 mlaqueous solution of MgSO₄.7H₂O to provide a cation to tigecycline molarratio of 5:1. The pH of the solution is adjusted by adding NaOH to a pHin the range of pH 6.0-pH 7.0. The solution is then lyophilized todryness. Reconstitution of the lyophile in 10 ml water results in asolution having a pH in the range of pH 6.0-pH 7.0.

Formulation 10

A formulation comprising tigecycline with MgSO₄ and NaOH suitable fortopical administration is prepared. 50 mg tigecycline is added to 10 mlaqueous solution of MgSO₄.7H₂O to provide a cation to tigecycline molarratio of 12:1. The pH of the solution is adjusted by adding NaOH to a pHin the range of pH 6.0-pH 7.0. The solution is then lyophilized todryness. Reconstitution of the lyophile in 10 ml water results in asolution having a pH in the range of pH 6.0-pH 7.0.

Formulation 11

A formulation comprising tigecycline with CaCl₂ and NaOH suitable fortopical administration is prepared. 50 mg tigecycline is added to 10 mlaqueous solution of CaCl₂.6H₂O to provide a cation to tigecycline molarratio of 5:1. The pH of the solution is adjusted by adding NaOH to a pHin the range of pH 6.0-pH 7.0. The solution is then lyophilized todryness. Reconstitution of the lyophile in 10 ml water results in asolution having a pH in the range of pH 6.0-pH 7.0.

Formulation 12

A formulation comprising tigecycline with CaCl₂ and NaOH suitable fortopical administration is prepared. 50 mg tigecycline is added to 10 mlaqueous solution of CaCl₂.6H₂O to provide a cation to tigecycline molarratio of 12:1. The pH of the solution is adjusted by adding NaOH to a pHin the range of pH 6.0-pH 7.0. The solution is then lyophilized todryness. Reconstitution of the lyophile in 10 ml water results in asolution having a pH in the range of pH 6.0-pH 7.0.

Example 14 Minocycline Kits

Kit 1

A kit is prepared comprising two vials. The first vial is prepared bydissolving 108 mg minocycline HCl in an acidic solution. The solution islyophilized to dryness. The second vial contains 10 ml diluent thatincludes 26.9 mg/ml MgSO₄.7H₂O and 13.6 mg/mL Na(C₂H₃O₂)₂.3H₂O. Thelyophile is then reconstituted with the diluent prior to use.

Kit 2

A kit is prepared comprising two vials. The first vial is prepared bydissolving 108 mg minocycline HCl in an acidic solution. The solution islyophilized to dryness. The second vial contains 10 ml diluent thatincludes 26.9 mg/ml MgSO₄.7H₂O and enough NaOH to adjust the pH toapproximately 5. The lyophile is then reconstituted with the diluentprior to use.

All references cited herein, including but not limited to published andunpublished applications, patents, and literature references, areincorporated herein by reference in their entirety and are hereby made apart of this specification. To the extent publications and patents orpatent applications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.The term “comprising” as used herein is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. Allnumbers expressing quantities of ingredients, reaction conditions, andso forth used in the specification are to be understood as beingmodified in all instances by the term “about.” Accordingly, unlessindicated to the contrary, the numerical parameters set forth herein areapproximations that may vary depending upon the desired propertiessought to be obtained. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of anyclaims in any application claiming priority to the present application,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

The above description discloses several methods and materials of thepresent invention. This invention is susceptible to modifications in themethods and materials, as well as alterations in the fabrication methodsand equipment. Such modifications will become apparent to those skilledin the art from a consideration of this disclosure or practice of theinvention disclosed herein. Consequently, it is not intended that thisinvention be limited to the specific embodiments disclosed herein, butthat it cover all modifications and alternatives coming within the truescope and spirit of the invention.

What is claimed is:
 1. A method of treating a bacterial infection,comprising administering a therapeutically effective amount of apharmaceutical composition to a subject in need thereof via topicaladministration to the eye of the subject, the pharmaceutical compositioncomprising an aqueous solution of tigecycline and a divalent ortrivalent metal cation, wherein the pH of the solution is greater than 4and less than 7 and the molar ratio of divalent or trivalent metalcation to tigecycline is greater than 6:1.
 2. The method of claim 1,wherein the molar ratio of divalent or trivalent metal cation totigecycline is greater than 8:1.
 3. The method of claim 1, wherein themolar ratio of divalent or trivalent metal cation to tigecycline isgreater than 10:1.
 4. The method of claim 1, wherein the molar ratio ofdivalent or trivalent metal cation to tigecycline is about 12:1.
 5. Themethod of claim 1, wherein the divalent or trivalent metal cation is adivalent metal cation.
 6. The method of claim 1, wherein the divalent ortrivalent metal cation is calcium.
 7. The method of claim 1, wherein thesolution comprises calcium chloride or calcium sulfate.
 8. The method ofclaim 1, wherein the divalent or trivalent metal cation is magnesium. 9.The method of claim 1, wherein the solution comprises magnesium sulfateor magnesium chloride.
 10. The method of claim 1, wherein theconcentration of tigecycline is at least 1 mg/ml.
 11. The method ofclaim 1, wherein the concentration of tigecycline is 5 mg/ml.
 12. Themethod of claim 1, wherein the solution comprises one or more additionalconstituents selected from the group consisting of hydrocortisone,dexamethasone, thonzonium bromide, tyloxapol, boric acid, andbenzalkonium chloride.
 13. The method of claim 1, wherein the solutiondoes not comprise a component selected from the group consisting of anantioxidant, a pyridine-containing compound, gluconate, an alcohol,glycerol, polyethylene glycol, a pyrrolidone-containing compound, awater-miscible local anaesthetic, urea, lactose, and a dehydratingagent.
 14. The method of claim 1, wherein the pharmaceutical compositionis contained within a vial prior to administration.
 15. The method ofclaim 14, wherein the vial is a single-use vial.
 16. A method oftreating a bacterial infection, comprising: reconstituting awater-soluble solid composition in a pharmaceutically acceptable diluentto form a solution; and administering a therapeutically effective amountof the solution to a subject in need thereof via topical administrationto the eye of the subject, wherein the water-soluble solid compositioncomprises tigecycline and a divalent or trivalent metal cation, whereinthe molar ratio of divalent or trivalent metal cation to tigecycline isgreater than 6:1.
 17. The method of claim 16, wherein the molar ratio ofdivalent or trivalent metal cation to tigecycline is greater than 8:1.18. The method of claim 16, wherein the molar ratio of divalent ortrivalent metal cation to tigecycline is greater than 10:1.
 19. Themethod of claim 16, wherein the molar ratio of divalent or trivalentmetal cation to tigecycline is about 12:1.
 20. The method of claim 16,wherein the divalent or trivalent metal cation is a divalent metalcation.
 21. The method of claim 16, wherein the divalent or trivalentmetal cation is calcium.
 22. The method of claim 16 comprising calciumchloride or calcium sulfate.
 23. The method of claim 16, wherein thedivalent or trivalent metal cation is magnesium.
 24. The method of claim16 comprising magnesium sulfate or magnesium chloride.
 25. The method ofclaim 16, wherein the water-soluble solid composition is in the form ofa lyophile.
 26. The method of claim 16, wherein the water-soluble solidcomposition is contained within a vial.
 27. The method of claim 26,wherein the vial is a single-use vial.
 28. The method of claim 26,wherein the vial comprises greater than 30 mg and less than 100 mg oftigecycline.
 29. The method of claim 26, wherein the vial comprises atleast 50 mg of tigecycline.
 30. The method of claim 26, wherein the vialcomprises at least 200 μg of tigecycline.
 31. The method of claim 26,wherein the vial comprises at least 1 mg of tigecycline.
 32. The methodof claim 16, wherein the pharmaceutically acceptable diluent is water.